Expression of surface layer proteins

ABSTRACT

A host cell which is provided with a S-layer comprising a fusion polypeptide consisting essentially of: 
     (a) at least sufficient of a S-layer protein for a S-layer composed thereof to assemble, and 
     (b) a heterologous polypeptide which is fused to either the carboxy terminus of (a) or the amino terminus of (a) and which is thereby presented on the outer surface of the said cell; can be used as a vaccine, for screening for proteins and antigens and as a support for immobilizing an enzyme, peptide or antigen. A process of transforming B. Sphaericus cells comprising electroporation is also provided.

This application is a national stage application of PCT/EP95/00147.

The present invention relates to vaccines and proteins, rDNA moleculesencoding protein expression and presentation systems for the productionand presentation of the said proteins, expression vectors therefor, andhosts transformed therewith, as well as methods involved therewith.

The traditional view on the native polymeric organization of thebacterial cell wall has changed dramatically over recent years with thedevelopment of new techniques for electron microscopic analysis. Theclassical idea that the cell membrane(s) is(are) covered by apeptide-glycan-containing matrix does not hold any longer. Besidesadditional surface structures such as capsules, sheaths, slimes orfimbriae, proteinaceous surface arrays or S-layers are being recognizedas a main constituent of the bacterial cell wall (Sleytr and Messner,1988).

S-layers are a common feature in archaebacterial surfaces (Konig, 1988).In some species such as Halobacterium salinarum or Thermoproteus spp.the proteinaceous S-layer even forms the sole cell wall. At presentS-layers are being detected with increasing frequency in a large rangeof gram-positive and gram-negative eubacteria. Surface arrays arecomposed of protein or glycoprotein subunits that are arranged into aparacrystalline two-dimensional array, displaying hexagonal, tetragonalor oblique symmetry. Self-assembly of the S-layer is an inherentproperty of the subunit and is the result of non-covalentprotein-protein interactions mediated through salt bridging by divalentmetal cations (Mg²⁺ or Ca²⁺). Non-covalent interactions with componentsof the underlying cell envelope are thought to be responsible for itspositioning at the outermost surface.

Despite the cloning and the characterization of several genes encodingS-layer proteins (SLP's), their function still remains speculative. Avariety of functions have been attributed to surface arrays. They mightserve as a protective barrier against degradative enzymes or predators,such as Bdellovibrio or help in maintaining bacterial cell shape andform. In some bacterial pathogens, S-layers have been identified asimportant virulence factors. Although S-layers have several physicalfeatures in common, general conclusions on their function cannot yetbeen drawn.

SLP's are thus present in a large number of archaebacteria, as well asgram-positive and, to a lesser extent, gram-negative bacteria. SLP'sform a main constituent of the cell wall, being capable of self-assemblyinto arrays (crystalline arrays) at the outermost surface of the cellwall. SLP's are continuously and spontaneously produced in largeramounts than any other class of protein in the cell.

SLP's are expressed and either presented or secreted by systems thereforwithin cells. The genes of these SLP system(s) include: strong promotersequence(s), a signal peptide coding sequence which is locateddownstream of the promoter sequence(s), a SLP coding sequence and atranscription termination sequence. The SLP coding sequence is locateddownstream from the signal peptide coding sequence, having its5'-terminus operatively linked to the 3'-terminus of the signal peptidecoding sequence.

As described herein, an SLP presentation system is distinguished from anSLP secretion system. In the former, the SLP's are bound-up in the cellwall of a host where they are thus presented. In the latter, the SLP'sare either produced in the cytoplasm (intracellular production) orsecreted into the surrounding medium (extracellular secretion).

The SLP expression and secretion systems of several bacteria have beenwell-characterized. Among these are those SLP expression and secretionsystems of bacteria of the genus Bacillus. Bacilli are well-known asabundant producers of SLP's.

More particularly, the SLP expression and secretion system of thespecies Bacillus brevis has been extensively studied for its potentialuse in expressing and extracellularly secreting large quantities ofpredetermined proteins. B. brevis is able to secrete large amounts ofextracellular SLP which are used to aid translocation of thepredetermined protein across B. brevis's unique two-layer cell wall forextracellular secretion thereof. Also, B. brevis does not secreteextracellular proteases in quantities which may degrade and inactivatethe extracellularly-produced proteins.

Tsukagoshi et al (1985) discloses the fusion of the α-amylase gene ofBacillus stearothermophilius DY-5 to the SLP coding gene of B. brevis47-5 for the expression of α-amylase in B. stearothermophilius DY-5, B.brevis 47-5, Escherichia coli HB101 and Bacillus subtilis 1A289 hoststhat are transformed therewith. Comparison studies showed that the B.brevis secretion levels were one hundred (100) times higher than that ofB. stearothermophilius itself. B. brevis secretion levels were fifteen(15) times higher than those of E. coli and five (5) times higher thanthose of B. subtilis. The efficient secretion of the enzyme in B. brevisis suggested therein as being due to the unique properties of the cellwall of the B. brevis.

Yamagata et al (1987) discloses the translational fusion of the5'-region of the gene coding for the middle wall protein (a SLPparticular to B. brevis) of B. brevis 47 with the α-amylase gene ofBacillus licheniformis for expression in B. brevis 47. The translationalfusion of these genes is reported as achieving efficient levels ofα-amylase production in B. brevis 47.

Tsukagoshi (1987/8) discloses the translational fusion of the genecoding for swine pepsinogen with the 5'-region of the middle wallprotein gene of B. brevis for expression in B. brevis 47 and B. brevisHPD31. Translational fusion of the 5'-region to the CGTase gene ofBacillus macerans also resulted in the expression of the efficientlevels of CGTase in B. brevis 47.

EP-A-0257189 in the name of Higeta Shoyu Co., Ltd., et al., discloses aseries of B. brevis strains which may be utilised as hosts to producelarge amounts of proteins without producing deleterious amounts ofextracellular proteases.

GB-A-2182664 in the name of Udaka discloses "expressing genes" that arederived from B. brevis 47 and which may be fused to genes coding forheterologous proteins. Among the heterologous genes suggested as beingappropriate for being fused to the genes derived from B. brevis 47 arevarious eucaryotic genes (such as those genes coding for interferon andinsulin) as well as procaryotic genes (such as those genes coding fortryptophanase and aspartate ammonia lyase). The fused genes may then beincorporated into expression vectors for transforming B. brevis 47.

Adachi et al (1989) discloses the fusion of the co-transcriptional cellwall protein (cwp) gene operon (coding for both the middle wall proteinand the outer wall protein) of B. brevis 47 with the gene coding forα-amylase in B. licheniformis in order to provide extracellularproduction of B licheniformis α-amylase by B. brevis 47 and B. subtilisIA289. The presence of several different cwp operon transcripts and thepresence of at least three different promoters (referred as therein asthe P1, P2 and P3 promoters) were confirmed. It was reported that the P1and P3 promoters were used in the same extent in B. brevis and B.subtilis, whereas the P2 promoter was reported to be used much lessfrequently in B. subtilis than in B. brevis.

Takao et al (1989) discloses an expression-secretion vector fortransforming B. brevis hosts for producing heterologous proteins,including eucaryotic proteins, such as swine pepinsogen. The vectorutilizes the promoter, the signal-peptide coding sequences and nine (9)amino-terminal amino acids of a middle wall protein of B. brevis whichare fused to a heterologous protein coding sequence. The hoststransformed thereby are B. brevis 47 and HPD31.

Yamagata et al (1989) discloses a host-vector system utilizing strains(47 and HPD31) of B. brevis that hyperproduce SLP's as the hosts.Expression-secretion vectors are constructed from multiple promoters,the peptide-signal coding region and a structural gene for one of themajor cell wall proteins of B. brevis 47. The B. brevis 47 genes werefused to a synthetic gene coding for human epidermal growth factor(hEGF).

In addition to the use of SLP expression and secretion systems derivedfrom B. brevis in B. brevis, it has also been disclosed to utilize SLPexpression and secretion systems of B. brevis in B. subtilis hosts.Tsuboi et al (1989) discloses the transformation of B. subtilis withgenes from B. brevis 47 that code for middle wall proteins. Thetransformed B. subtilis is thus capable of expressing the middle wallprotein of B. brevis.

It has also been disclosed by Tang et al (1989) that the SLP expressionand secretion system of an alkaline phosphatase secretion-deficientmutant strain (strain NM 105) of B. licheniformis 749/C can be clonedinto mutant strains of both E. coli (strain NM 539) and B. subtilis(strain MI112). Bowditch et al 1989 discloses cloning the gene codingfor the SLP of B. sphaerius into E. coli TB1, JM101 and JM107. Thetransformed E. coli hosts then expressed the B. sphaericus SLP.

Lucas et al (1994), while studying the S-layer protein of Acetogeniumkivui, disclose that there exists a repeated peptide sequence at theN-terminus of said S-layer protein which is shared by several differentS-layer proteins, such as the middle wall protein from B. brevis and theS-layer protein form B. sphaericus 2362, and these authors suggest thatthis conserved domain is essential to anchor these S-layer proteins tothe underlying peptidoglucan. Interestingly, Matuschek et al. (1994)disclose that the same conserved domain, which is found at theN-terminus of the Acetogenium kivui S-layer, is also present in thesequence of the extracellular, cell-bound pullulanase fromThermoanaerobacterium thermosulfurigenes, but in the latter case it islocated near the C-terminus of the polypeptide.

U.S. Pat. No. 5,043,158 discloses pharmaceutical compositions whichcomprise carriers that are chemically-coupled to epitope-bearingmoieties. The carriers are isolated crystalline or paracrystallineglycoproteins, especially those derived from the SLP's of Clostridiumthermohydrosulfuricum and B. stearothermophilus. The conjugates formedthereby were reported as being capable of eliciting the formation ofantibodies as well as eliciting B-cell mediated and T-cell mediatedresponses.

It is a primary objective of the present invention to provide arecombinant DNA (rDNA) molecule that includes a SLP system capable ofexpressing and presenting, rather than expressing and secreting, afusion polypeptide (such as a fused SLP/antigenic peptide) in a widevariety of bacteria including bacteria of the genus Bacillus and, moreparticularly, B. sphaericus.

It is yet another primary object of the present invention to providesuch a rDNA molecule which includes, derived from B. sphaericus, SLPpromoter sequence(s), a SLP signal-peptide coding sequence and a SLPcoding sequence which codes for at least a functional portion of thesurface layer protein of B. sphaericus and which may be fused to aheterologous coding sequence coding for a heterologous polypeptide (suchas an antigenic peptide), such that the expression of the heterologouspolypeptide is placed under the control of the said promoter(s) andfurther such that the heterologous polypeptide expressed thereby will befused to the SLP so as to be bound-up in the cell wall of the host forpresentation thereof on the outer surface of the host's cell wall foreliciting an immunogenic response thereto.

It is a yet further primary object of the present invention to providevectors containing such rDNA molecules, which vectors may be used toeffectively transform host cells.

It is a still yet further primary object of the present invention toprovide hosts, especially hosts of the genus Bacillus, and moreparticularly of the species B. sphaericus, which are transformed withvectors containing such rDNA molecules, which express fusionpolypeptides (such as antigenic peptides) produced thereby and whichpresent the expressed fusion polypeptides for, inter alia, eliciting animmunogenic response thereto.

A still further primary object of the present invention is to providemethods for forming the rDNA molecules, for preparing the appropriatevectors therefor, for transforming hosts with such vectors and forproducing the fusion peptides (vaccines and proteins) of the presentinvention.

The present invention provides a host cell which is provided with aS-layer comprising a fusion polypeptide consisting essentially of:

(a) at least sufficient of a SLP for a S-layer composed thereof toassemble, and

(b) a heterologous polypeptide which is fused to either the carboxyterminus of (a) or the amino terminus of (a) and which is therebypresented on the outer surface of the said cell.

Preferably, the heterologous polypeptide is fused to the carboxyterminus of (a).

The amount of a SLP which is sufficient in its own right for a S-layercomposed thereof to form is termed herein the "functional portion" ofthe SLP. The fusion polypeptide thus typically incorporates at least thefunctional portion of a SLP native to the host cell. Sacculi derivedfrom a host cell according to the invention also form part of theinvention.

The heterologous polypeptide may be an antigenic peptide. In that event,the invention provides a vaccine comprising a host cell or sacculiaccording to the invention wherein the heterologous polypeptide is anantigenic peptide and a pharmaceutically or veterinarily acceptablecarrier or diluent.

The invention further provides a recombinant DNA molecule whichcomprises a promoter operably linked to a coding sequence which encodesa signal peptide and a fusion polypeptide, the signal peptide beingcapable of directing the said fusion polypeptide to be presented on thesurface of a host cell in which expression occurs and the fusionpolypeptide consisting essentially of a heterologous polypeptide fusedto either the carboxy terminus or to the amino terminus of at leastsufficient of a SLP for a S-layer composed thereof to assemble.

An efficient and reliable system which employs an SLP expression andpresentation system is therefore provided for the expression andpresentation of fusion polypeptides (such as antigenic peptides forvaccines) in a wide variety of, preferably, Bacilli. This systemincludes a recombinant DNA molecule having a promoter that is fused to afunctional DNA sequence, so that the functional DNA sequence is placedunder the control of the promoter. The functional DNA sequence includesa SLP coding sequence which codes for at least a functional portion of aSLP. The functional DNA sequence further includes a heterologouspolypeptide coding sequence that codes for a heterologous polypeptide(such as an antigenic peptide for use as a vaccine or a protein) whichpeptide coding sequence is fused to the SLP coding sequence.

As used herein, the term "functional DNA sequence" refers to DNAsequences that contain all of the various sequences (with the exceptionof the promoter sequence), both coding (such as sequences coding for theproteins whose expression and presentation is desired) and non-coding(such as control sequences and regulatory regions, i.e. sequences thatare necessary or desirable for the transcription and translation of acoding sequence to which they are operably linked or fused when they arecompatible with the host into which they are placed) which, whenoperably joined (by linking, fusing or otherwise) to a promoter andplaced into a compatible host, permit the sequence to be operational andexpress and present the protein(s) coded for by the coding sequence(s)thereof.

As used herein, the terms "presented", "presentation", "present" and/or"presents" refer to the manner in which the heterologous polypeptide(for example an antigenic peptide or protein) is positioned whenprovided as part of a hybrid particle (such as the fusion vaccine orfusion protein) in such a way as to elicit an immune response to theheterologous polypeptide.

"Presentation systems" are DNA sequences which include both a codingsequence coding for a polypeptide (such as a heterologous polypeptide)whose presentation is desired, and other appropriate sequences thereforewhich permit such presentation when the DNA sequences are compatiblewith the host into which they are placed.

"Expressions systems" are DNA sequences which include both a codingsequence coding for polypeptide(s) whose expression is desired andappropriate control sequences therefor which permits such expressionwhen the DNA sequences are compatible with the host into which they areplaced. As is generally understood, "control sequences" refers to DNAsegments which are required for, or which regulate, expression of thecoding sequence with which they are operably joined.

We have found that it is the amino-terminal portion of a SLP which issufficient for S-layer formation. We have found that more than the first19.56% by number of the amino acid residues, as measured from theamino-terminus of a mature SLP, are required for a S-layer to form. By a"mature SLP" we mean a SLP without signal peptide residues. More thanthe N-terminal 239 amino acid residues of the mature SLP shown in FIG. 6are thus required. The first 41.41% by number of the amino acid residuesof a mature SLP are sufficient, for example the first 506 amino acidresidues of the mature SLP shown in FIG. 6.

More than the first 20% amino acid residues of an active SLP may bepresent. The SLP portion of a fusion polypeptide may therefore consistof the first N-terminal 28% or more or 35% or more, for example the mostN- terminal 41% or more or 50% or more or 60% or more or 80% or more, oreven all, amino acid residues of a mature SLP such as the mature SLPshown in FIG. 6. From the first N-terminal 28% to all, for example fromthe first N-terminal 35% or 41% or 50% or 60% or 80% to all, of theamino acid residues of a mature SLP can be present. The first N-terminal400 or more, 600 or more, 800 or more or 1000 or more amino acidresidues of a mature SLP may be present. A convenient restriction sitein the DNA coding sequence of a SLP will typically determine theC-terminus of the SLP portion of a fusion polypeptide.

The SLP portion of a fusion polypeptide is typically homologous withrespect to the host cell in which the fusion polypeptide is expressed.In other words, the portion of the SLP which is present in the fusionpolypeptide should generally be from a SLP of the same species as thehost in which the fusion polypeptide is expressed. Typically the portionof the SLP in the fusion polypeptide is from the native SLP of the hostin which the fusion polypeptide is expressed. The fusion polypeptide mayincorporate an appropriate portion of a SLP of a bacterium of the genusBacillus, for example of the species B. brevis or B. sphaericus.

The heterologous polypeptide may be a physiologically active polypeptideor a foreign epitope (an antigenic determinant, peptide immunogen orepitope-bearing moiety, as shall be discussed at greater length below).The carboxy terminus of the functional portion of a SLP may be fuseddirectly to the amino terminus of the physiologically active polypeptideor the foreign epitope. The fusion polypeptide therefore may consistessentially of the functional portion of a SLP and, fused directly tothe carboxy terminus thereof, a heterologous amino acid sequence.Alternatively, the amino terminus of the functional portion of a SLP maybe fused directly to the carboxy terminus of the physiologically activepolypeptide or the foreign epitope. The fusion polypeptide therefore mayconsist essentially of the functional portion of a SLP and, fuseddirectly to the amino terminus thereof, a heterologous amino acidsequence.

Alternatively, an intervening linker sequence may be present between thefunctional portion of the SLP and the heterologous polypeptide. Thelinker sequence may be from 1 to 20, for example, from 1 to 5 or from 1to 10 amino acid residues long. The linker sequence may be designed toincorporate a cleavage site recognized by cyanogen bromide or a cleavageenzyme.

The heterologous polypeptide is a polypeptide whose expression is notnormally controlled by a SLP promoter i.e. is not a naturally occurringSLP. The heterologous polypeptide can be a physiologically activepolypeptide such as an enzyme. The polypeptide may be a polypeptide drugor a cytokine. Specific polypeptides which may be mentioned areα-amylase, tissue plasminogen activator, luteinizing hormone releasinghormone, a growth hormone such as human growth hormone, insulin,erythropoietin, an interferon such as α-interferon, and calcitonin.

Alternatively, the heterologous polypeptide may comprise a foreignepitope or polypeptide immunogen. The polypeptide immunogen thereforetypically comprises an antigenic determinant of a pathogenic organism.The immunogen can be an antigen of a pathogen. The pathogen may be avirus, bacterium, fungus, yeast or parasite. The foreign epitope may bean epitope capable of inducing neutralizing or non-neutralizing antibodyor of inducing a cellular immune response.

The immunogen or epitope may be derived from a virus such as a humanimmunodeficiency virus (HIV) such as HIV-1 or HIV-2; a hepatitis virussuch as hepatitis A, B or C; a poliovirus such as poliovirus type 1, 2or 3; influenza virus; rabies virus; or measles virus. Examples ofbacteria from which an immunogen or epitope may be derived include B.pertussis, C. tetani, V. cholera, N. meningitides, N. gonorrhoea, C.trachomatis and E. coli. The immunogen may therefore be the P69 antigenof B. pertussis, pertussis toxin or a subunit thereof, tetanus toxinfragment C, E. coli heat labile toxin B subunit (LT-B) or an E. coli K88antigen, or an antigenic portion thereof. An immunogen derived from aparasite may be an immunogen derived from P. falciparum, a causativeagent of malaria.

As used herein, the terms "antigenic peptide", "antigenic determinant","peptide immunogen", "polypeptide immunogen", "epitope" and"epitope-bearing moiety" all refer to substances that contain a specificdeterminant which induces an immune response (such as the production ofantibodies or the elicitation of T-cell mediated response). Thesubstance may itself be a hapten (i.e. a simple moiety which, whenrendered immunogenic, behaves as an antigen) or it may be a more complexmoiety, only portions of which are responsible for immunospecificitywith regard to the antibodies obtained.

As used herein, the terms "immunogenic response" and "immune response"refer to the biological responses, such as the raising of antibodies orthe elicitation of T-cell or B-cell mediated responses, that areelicited in an organism (such as a mammal) by the presence of an antigenor immunogen.

The present invention also provides recombinant DNA vectors comprising arecombinant DNA molecule according to the present invention and furtherprovides a host cell transformed with such a recombinant DNA vector. Thevector is typically an expression vector. The fusion polypeptide canthereby be expressed in a suitable host cell transformed with such anexpression vector. A S-layer composed of the fusion polypeptide that isexpressed can thereby be assembled on the surface of the host cell.

An expression vector can include any suitable origin of replicationwhich will enable the vector to replicate in a bacterium. A ribosomebinding site is provided. The ribosome binding site is suitably locatedbetween the promoter and the DNA sequence encoding the heterologouspolypeptide. If desired, a selectable marker gene such as an antibioticresistance gene can be provided in the vector. The vector is generally aplasmid.

The vector is normally provided with a transcriptional terminationsequence. The coding sequences of the recombinant DNA molecules andvectors of the invention are provided with translational start and stopcodons. Vectors may be constructed by assembling all appropriateelements using techniques known in the art (Sambrook et al. 1989).

According to the invention, a host cell provided with a S-layercomprising a fusion polypeptide is prepared by a process whichcomprises:

(i) providing a suitable host cell incorporating a recombinant DNAmolecule which comprises a promoter operably linked to a coding sequencewhich encodes a signal peptide and a fusion polypeptide, the signalpeptide being capable of directing the said fusion polypeptide to bepresented on the surface of the said host cell and the fusionpolypeptide consisting essentially of a heterologous polypeptide fusedto either the carboxy terminus or the amino terminus of at leastsufficient of portion of a S-layer protein for a S-layer composedthereof to assemble on the surface of the said host cell; and

(ii) culturing the said host cell so that the said fusion polypeptide isexpressed and a S-layer comprising the fusion polypeptide is formed onthe surface of the said host cell, the heterologous polypeptide therebybeing presented on the outer surface of the said host cell.

In a preferred variant of the invention, a host cell provided with aS-layer comprising a fusion polypeptide is prepared by a process whichcomprises:

(i) providing a suitable host cell incorporating a recombinant DNAmolecule which comprises a promoter operably linked to a coding sequencewhich encodes a signal peptide and a fusion polypeptide, the signalpeptide being capable of directing the said fusion polypeptide to bepresented on the surface of the said host cell and the fusionpolypeptide consisting essentially of a heterologous polypeptide fusedto the carboxy terminus of at least sufficient of the amino terminalportion of a S-layer protein for a S-layer composed thereof to assembleon the surface of the said host cell; and

(ii) culturing the said host cell so that the said fusion polypeptide isexpressed and a S-layer comprising the fusion polypeptide is formed onthe surface of the said host cell, the heterologous polypeptide therebybeing presented on the outer surface of the said host cell.

Preferably the host cell is one which does not secrete extracellularproteases. The host cell is generally a bacterium, typically a bacteriumwhich naturally produces a S-layer protein, i.e. a bacterium which inits native state has a S-layer on its surface. Depending upon theintended use, the bacterium may be a gram-positive or gram-negativebacterium. Host bacteria include bacteria of the genera Cocci andBacilli may be transformed, for example Staphylococcus, Streptococcus,Corynebacterium, Lactobacillus, Bacillus, Clostridium and Listeria.Preferably, host bacteria include bacteria of the genera Bacilli. Usefulbacteria in which the present invention may be applied are thereforeBacillus sphaericus and B. brevis.

Bacillus sphaericus is a bacterium of the genus Bacillus in which asubstantial quantity of the SLP's produced thereby are bound-up in theS-layer of the cell wall thereof and are not secreted extracellularly.As such, unlike B. brevis and B. subtilis, we have found that B.sphaericus possesses what is potentially an efficient SLP presentationsystem.

The structure and properties of B. sphaericus have been characterized(see, for example Lewis et al (1987); Howard et al (1973) and Lepault etal (1986)). B. sphaericus (like the other Bacilli) has a high level ofgrowth throughout its growth cycle, thereby increasing the quantities ofthe fusion polypeptide that can be expressed and presented thereby.

A preferred strain of B. sphaericus is B. sphaericus P-1. B. sphaericusP-1 has been deposited under the Budapest Treaty of the BelgianCoordinated Collections of Microorganisms (BCCM), LMG CultureCollection, Universiteit Gent, Lab. voor Microbiologie, K.L.Ledeganckstraat 35, B-9000 Gent, Belgium. The deposit was made on 13thMay 1993 and was given accession number LMG P-13855. B. sphaericus P-1offers the further advantage of not producing detectable levels ofextracellular proteases which can cause damage to fusing polypeptidesproduced according to the invention.

The signal peptide is typically a signal peptide for a SLP, for examplefor a SLP of a bacterium of the genus Bacillus. It may be a signalpeptide for a SLP of B. brevis, B. sphaericus or B. subtilis for exampleB. sphaericus P-1. Preferably the signal peptide is the signal peptidefor the SLP of which an appropriate portion is incorporated in thefusion polypeptide. A signal peptide which is homologous, i.e. which isderived from the same species of cell, with respect to the host cell inwhich expression of the fusion polypeptide is to occur can be employed.Preferably the native signal peptide for the native SLP of the host cellin which expression is to occur is provided.

A useful process for preparing a host cell provided with a S-layercomprising a fusion polypeptide comprises:

(a) providing an intermediate vector in which the coding sequence for aninternal portion of the native SLP of the said host cell hastranslationally fused to the 3'-end thereof the coding sequence for theheterologous polypeptide and in which the said coding sequences areprovided upstream of a promotorless selectable marker gene such thatthey form a translational or transcriptional fusion therewith;

(b) transforming the said host cell with the intermediate vector;

(c) selecting a transformed host cell which has a S-layer comprising thesaid fusion polypeptide.

This process relies upon the occurrence of a single homologousrecombination as a result of the introduction of the intermediate vectorinto the host cell. The intermediate vector is typically a plasmid. Aninternal portion of the native SLP lacks the amino-terminal andcarboxy-terminal amino acid residues of the native SLP. Up to the first50, up to the first 100, up to the first 200, up to the first 300, up tothe first 400 or up to the first 500 of the amino-terminal amino acidresidues may be missing. Independently up to the first 50, up to thefirst 100, up to the first 200, up to the first 300, up to the first 400or up to the first 500 carboxy-terminal amino acid residues may bemissing.

The coding sequence for an internal portion of the native SLP thereforecorresponds to the native SLP gene lacking its 5'- and 3'-ends. Thiscoding sequence can be fused directly or via a sequence encoding alinker to the 5'- end of the coding sequence for the heterologouspeptide. Suitable linkers are described above. The promoterlessselectable marker gene may be the neomycin phosphotransferase II (nptII)gene which confers resistance to the antibiotics neomycin and kanamycin.The intermediate vector typically also comprises an origin ofreplication and a second selectable marker gene, for example anantibiotic resistance gene such as an erythromycin resistance gene.

In one preferred embodiment, therefore, a host cell having a S-layercomprising a fusion polypeptide can be prepared by the followingprocedure:

1. An appropriate intermediate vector is constructed that has followingcharacteristics:

the cloned part of the SLP in the vector has to be internal to the SLPgene, i.e. contain no borders of the gene;

the cloned part of the SLP gene is translationally fused to the sequenceencoding the heterologous peptide of interest;

both are cloned upstream of a promotorless first selectable marker gene(e.g. the nptII gene) so that they make a translational ortranscriptional fusion;

optionally a replicon (such as that of pIL253 for B. sphaericus) and/ora second selectable marker gene (such as the erythromycin (Em)resistance gene).

2. The intermediate vector is introduced into an appropriate host cellsuch as B. sphaericus P-1, for example via electroporation.

3. Transformants are selected by means of the second selectable marker,for example Em resistant transformants selected. This can enable thestructure of the intermediate vector to be verified.

4. The selected transformant(s) are grown, for example overnight in LBmedium containing 10 μg/ml Em.

5. The transformants thus grown are plated out. For example a bacterialsuspension can be plated out directly on LB+ agar containing 5-10 μg/mlneomycin (Nm) when the first selectable marker gene is the nptII gene ordilute starter culture in LB liquid medium containing the same amount ofNm, again when the first selectable marker gene is the nptII gene.

6. Colonies are selected by means of the first selectable marker, forexample single Nm resistant colonies.

7. Occurrence of a single homologous recombination is verified, forexample by Southern analysis.

8. Formation of the recombinant fusion polypeptide is verified, forexample by sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE).

Alternatively, a host cell provided with a S-layer comprising a fusionpolypeptide can be produced by a process comprising:

(a) fusing to a promoter a SLP coding sequence coding for the signalpeptide and at least sufficient of the amino-terminal portion of a SLPfor a S-layer composed thereof to assemble on the surface of the hostcell, and fusing a peptide coding sequence coding for the heterologouspolypeptide to the 3'-end of the SLP coding sequence, whereby arecombinant DNA molecule for the expression and presentation of thefusion polypeptide is prepared;

(b) inserting the recombinant DNA molecule into a suitable vector,whereby a recombinant DNA vector is prepared;

(c) transforming a suitable host cell with the recombinant DNA vector,whereby a transformed host cell having the recombinant DNA molecule isprovided;

(d) culturing the transformed host cell, whereby the fusion polypeptideis expressed and a S-layer comprising the fusion polypeptide isassembled on the host cell wall.

The transformed host cell is thus cultured in an appropriate culturemedium. As a result of expression and presentation of the fusionpolypeptide on the outer surface of the host cell, the heterologouspolypeptide is thus presented so that an immunogenic response can bestimulated thereto when the host cell is administered to a human oranimal host. The S-layer will also comprise the host cell's native SLPunless steps are taken to disable production of that SLP.

DNA sequences consisting essentially of the appropriate coding sequencesmay be produced by ligation. The DNA sequences may be isolated and/orpurified for use in the invention. Expression vectors can thus beprepared which incorporate a promoter operably linked to one of theseDNA sequences. These vectors are capable of expressing the fusionpolypeptide when provided in a suitable host. The vectors are generallyplasmids.

The coding sequences are located between translation start and stopsignals. A ribosome binding site, an origin of replication and,optionally, a selectable marker gene such as an antibiotic resistancegene are typically present. In addition to the promoter, otherappropriate transcriptional control elements are provided, in particulara transcriptional termination site. The promoter may be the naturalpromoter for a SLP protein such as a promoter for a Bacillus SLP, forexample for a SLP from B. sphaericus or B. brevis. Typically thepromoter is the native promoter for the SLP at least the functionalportion of which is present in a fusion polypeptide according to theinvention. The coding sequences are provided in the correct frame suchas to enable expression of the fusion polypeptide to occur in a hostcompatible with the vector.

Transformation of a host cell may be achieved by conventionalmethodologies. We have found, however, that such methodologies do notwork in the case of B. sphaericus P-1. We have devised a new techniquefor transforming B. sphaericus P-1. Accordingly, the present inventionprovides a process of transforming B. sphaericus P-1 cells with DNA,which process comprises harvesting B. sphaericus P-1 cells at the latestationary growth phase, mixing the harvested cells with the DNA andeffecting electroporation to cause entry of the DNA into the said cells.

Electroporation at the late stationary phase may be effected at from 8to 16 kV/cm, 150 to 250 Ω and 20 to 40 μF. Preferred conditions are12kV/cm, 200 Ω and 25 μF. Electroporation is generally carried out inelectrocurvettes, for example 0.1 cm- or 0.2 cm-gapped electrocurvettes.

The transformed host cells are cultured under such conditions thatexpression of the fusion polypeptide occurs. The invention consequentlyadditionally provides a host cell transformed with a recombinant DNAmolecule, typically a vector, according to the invention.

The host cell can be transformed so that none of the native SLP is stillproduced or so that the native SLP is produced in addition to the fusionpolypeptide according to the invention. The invention therefore furtherprovides a host cell which is able to express a fusion polypeptideaccording to the invention in addition to or instead of he SLP native tothe said host cell.

A host cell can therefore be engineered which presents a foreign epitopeon its surface as a part of a composite S-layer. The S-layerincorporates the fusion polypeptide. The fusion polypeptide (forexample, presenting a foreign epitope) and any native SLP produced bythe host cell assemble into a S-layer. We have surprisingly found thatfusion of a foreign amino acid sequence to at least a functional portionof a S-layer protein does not prevent the proper folding of the foreignsequence. The foreign sequence is thus presented on the surface of hostcell and can be recognised by the immune system of a host, human oranimal.

The foreign sequence can also be presented on the surface of sacculi.Sacculi, sometimes termed native sacculi or ghosts, are devoid ofcytosolic and membrane proteins. They consist mainly of thepeptido-glycan outer layer of bacterial cells surrounded by the S-layer.They can be derived from host cells according to the invention by simpleprocedures (Saara and Sleytr, 1987). For example, host cells may besonicated, a detergent such as Triton X-100 added and the mixtureincubated. After washing, the treated cells can then be incubated withDNAse and RNAse. The resulting sacculi are washed again.

The host cell or sacculi derived therefrom can therefore be used as avaccine. The host cell may be a non-pathogenic bacterium. It may be abacterium which is naturally non-pathogenic or it may be an attenuatedbacterium for this purpose, i.e. an attenuated form of a pathogenicbacterium. An attenuated bacterium typically contains one or morerationally directed mutations that prevent extensive spreading of thebacterium within the host to which the bacterium is administered. Thebacterium can however still establish a limited infection leading to thestimulation of a natural immune response (Charles and Dougan, 1990).

A pharmaceutical or veterinary composition may therefore be providedwhich comprises a host cell provided with a S-layer comprising a fusionpolypeptide according to the invention and a pharmaceutically orveterinarily acceptable carrier or diluent. The composition may beformulated as a vaccine. The composition may be administered orally,intranasally or parenterally such as subcutaneously or intramuscularly.The dosage employed depends on a number of factors including the purposeof administration and the condition of the patient. When the host cellis a bacterium, typically however a dose of from 10⁹ to 10¹¹ bacteria issuitable for a human or animal for each route of administration.

The composition may be in lyophilized form. The composition may beformulated in capsular form. The capsules may have an enteric coatingfor oral administration, comprising for example Eudragate "S", Eudragate"L", cellulose acetate, cellulose phthalate or hydroxypropylmethylcellulose. These capsules may be used as such or alternatively, thelyophilised material may be reconstituted prior to administration, e.g.as a suspension. Reconstitution is advantageously effected in a bufferat a suitable pH to ensure the viability of the organisms. In order toprotect the bacteria from gastric acidity, a sodium bicarbonatepreparation is advantageously administered before each administration ofthe composition.

The presentation system of the invention has applicability beyond use aslive bacterial vaccines. The heterologous polypeptides which arepresented on the surface of host cells thus remain bound to the cells,so the presentation system may be used for screening proteins andantigens, and the system can also be used as a support for immobilisingan enzyme, peptide and/or antigen (Georgiou et al, 1993; Smith et al1993).

Host cells according to the invention may therefore be used for displayof antibodies and peptide libraries. A bacterial selection systemcomplementary to phage display technology can thus be produced. Thebacterial library can be separated by affinity chromatography.

A host cell displaying on its surface a heterologous polypeptide ofinterest can also be used to raise antibody against that polypeptide.Polyclonal antibody can be raised by, for example, administering thehost cell to a mammal. The mammal may be an experimental animal such asa rabbit, mouse or rat. Antisera can be obtained from the immunisedmammal.

Monoclonal antibodies can be obtained by adaptation of conventionalprocedures. A mammal is immunised with a host cell according to theinvention, cells of lymphoid origin from the immunised mammal are fusedwith cells of an immortalizing cell line and thus--immortalized cellswhich produce antibody specific for the heterologous polypeptide ofinterest are selected. The selected cells are cultured to obtainquantities of the desired monoclonal antibody.

In more detail, hybridoma cells producing monoclonal antibody may beprepared by fusing spleen cells from an immunised animal with a tumourcell. The mammal which is immunised may be a rat or mouse. Thehybridomas may be grown in culture or injected intraperitoneally forformation of ascites fluid or into the blood stream of an allogenic hostor immunocompromised host. Human antibody may be prepared by in vitroimmunisation of human lymphocytes with respect to the peptide or afragment thereof, followed by transformation of the lymphocytes withEpstein-Barr virus.

The presentation system of the invention can further be employed as awhole-cell adsorbent. The expression of a heterologous polypeptide aspart of the S-layer fusion polypeptide on the surface of host cellsenables the host cells to be employed as an affinity adsorbent. Hostcells may also be used to present an enzyme as the heterologouspolypeptide, thus acting as biocatalysts.

As a consequence of cloning and sequencing the gene encoding the SLP ofB. sphaericus P-1, in another aspect of the invention we have identifiedthree promoters associated with the gene. One of these promoters iscapable of directing a three-fold higher expression level than thewild-type promoter. A putative promoter previously indicated by Bowditchet al (1989) was found incapable of directing expression.

The present invention therefore additionally provides a first promoterhaving a -35 region of the sequence TTGAAT and a -10 region of thesequence TATATT. The critical parts of promoters are believed to be the-35 and -10 regions (Watson et al, 1987). According to the numberingscheme used, the DNA nucleotide encoding the beginning of the mRNA chainis +1.

Typically there are 16 to 18 nucleotides between the -35 and -10regions. Preferably the intervening nucleotides (SEQ ID NO:1) areTTCGGAAAAGATAGTGT. A useful promoter has the sequence (SEQ ID NO:2)CTAAATTTATGTCCCAATGCTTGAATTTCGGAAAAGATAGTGT TATATTATTGT. The -35 and -10regions are underlined.

A promoter having -35 and -10 regions of the sequences TTGAAT andTATATT, respectively, is the promoter having a transcription initiationsite identified herein as P1 (see FIG. 10 of the accompanying drawings).This promoter is capable of directing expression at higher levels thanthe promoters having transcription initiation sites identified herein asP2 and P3 (FIG. 10) or than the entire wild type promoter sequence shownin FIG. 10 incorporating all of the three promoters. The P1 promoter isin fact three-fold stronger but only when used alone, i.e. whenseparated from the P2 and/or P3 promoters.

The invention also provides a second promoter having a -35 region of thesequence CTTGGTT and a -10 region of the sequence TATAAT. Typicallythere are 16 to 18 nucleotides between the two regions. Preferably theintervening nucleotides (SEQ ID NO:3) are ATTATTGAGAGTAAGG. A usefulpromoter has the sequence (SEQ ID NO:4)TCCAGAAAATGCTTGGTTATTATTGAGAGTAAGGTATAATAGGTA, the -35 and -10 regionsbeing underlined.

The invention additionally provides a third promoter having a -35 regionof the sequence ATTACGGGA and a -10 region of the sequence TTTAGT.Typically there are 16 to 18 nucleotides between the two regions.Preferably the intervening nucleotides (SEQ ID NO:5) areGTCTAATTAATTTTTGACAA. A useful promoter has the sequence (SEQ ID NO:6)AAAATATTACGGGGAGTCTTTAATTTTTGACAATTTAGTAACCAT, the -35 and -10 regionsbeing underlined.

The three promoters may be tandemly arranged, for example in the orderof the third promoter, the second promoter and the first promoter in the5' to 3' direction. This is the order in which the three promoters occurin the wild-type promoter of B. sphaericus P-1 shown in FIG. 10. UsefulDNA fragments incorporating the promoters according to the invention arethe following DNA sequences shown in FIG. 10, using the number systememployed in that Figure:

nucleotides 52 to 353;

nucleotides 1 to 353;

nucleotides 1 to 406; and

nucleotides 1 to 455.

The promoters can be used to direct expression of a heterologous proteinin a host, for example a bacterial host such as a gram-negative orgram-positive bacterium. Suitable host cells are therefore as describedabove. The invention therefore provides:

(a) an expression vector which comprises a promoter according to theinvention and a downstream cloning site into which a DNA sequenceencoding a heterologous protein may be cloned such that the promoter isoperably linked to the said sequence;

(b) an expression vector which comprises a promoter according to theinvention operably linked to a DNA sequence encoding a heterologousprotein; and

(c) a DNA fragment comprising a promoter according to the inventionoperably linked to a DNA sequence encoding a heterologous protein.

An expression vector (a) or (b) can include any suitable origin ofreplication which will enable the vector to replicate. A ribosomebinding site is provided. The ribosome binding site is suitably locatedbetween the promoter and the cloning site or the DNA sequence encodingthe heterologous protein as the case may be. If desired, a selectablemarker gene such as an antibiotic resistance gene can be provided in thevector. The vector is generally a plasmid.

The cloning site of vector (a) may be provided at a translational startcodon such as a NcoI site. Alternatively, no translational start codonmay be provided in the vector. In that event, the foreign gene to beinserted into the cloning site would need to be provided with such acodon. Typically the gene inserted into the cloning site is providedwith a translational stop codon.

Both vectors (a) and (b) are normally provided with a transcriptionaltermination sequence. The DNA sequences of vector (b) and of the DNAfragment mentioned above are provided with translational start and stopcodons. As in the case of vectors (a) and (b), the DNA fragment willtypically incorporate a ribosome binding site downstream of thepromoter. The DNA fragment may be single- or double- stranded, dependingon its purpose.

Vectors (a) and (b) may be constructed by assembling all appropriateelements using techniques known in the art (Maniatis et al, 1982). Forexample, vector (b) may be obtained by cloning a DNA sequence encoding aheterologous protein into vector (a) at the cloning site of that vectoror by cloning a DNA fragment (c) into an expression vector provided withan origin of replication. The cloning site of vector (a) may beintroduced by oligonucleotide-directed mutagenesis or polymerase chainreaction (PCR)-mediated site specific mutagenesis. The elements ofvectors (a) and (b) are operably linked. The recombinant DNA fragment(c) may be constructed by ligating a foreign gene to a promoter sequenceaccording to the invention.

The DNA sequence encoding a heterologous protein may be providedimmediately downstream of a DNA sequence encoding a signal peptideresponsible for polypeptide secretion which in turn may be providedimmediately downstream of the translational start codon. The signalpeptide-encoding DNA sequence may encode any signal peptide capable ofdirecting secretion of polypeptides from gram-positive bacterium.Typically the amino acid sequence of the signal peptide endsValAlaSerAla.

The heterologous protein may be a heterologous peptide as describedabove.

The following Examples illustrate the invention. In the accompanyingdrawings:

FIG. 1 shows the characterization of pGVP1.

A. Restriction map of pGVP1 for HindIII (1), PstI (2), SspI (3), thathave double-occurring restriction sites. The single-occurring sites areindicated on the outside of the map.

B. Identification of a circular single-stranded DNA molecule as areplication intermediate of pGVP1.

Panel 1.Ethidium bromide-stained 1% agarose gel. Lane A, non-digestedtotal DNA of B. sphaericus P-1; lane B, HhaI digested P-1 total DNA;lane C, S1 nuclease-treated P-1 total DNA; lane D, P-1 total DNA treatedwith T₄ DNA polymerase.

Panel 2.Hybridization between ³² P-labelled pGVP1 and a non-denaturedSouthern blot of the gel in panel 1 on a nitrocellulose membrane. Aspecific hybridization signal, corresponding to single-stranded DNA, wasobserved only in non-digested (lane A) and T₄ DNA polymerase-treated P-1DNA (lane D), but not in HhaI (lane B) or S1 nuclease-treated total P-1DNA (lane C).

Panel 3.Hybridization between ³² P-labelled pGVP1 and a Southern blot ofa similar gel as in panel 1, but denatured prior to transfer to anitrocellulose membrane. In all lanes, a hybridization signal,corresponding to double-stranded DNA can be observed. In lanes A and D,additionally the signal corresponding to single-stranded DNA can beobserved.

FIG. 2 shows the results of plasmid analysis of Em-resistant B.sphaericus P-1 transformed by pIL253. Central panel. HindIII-digestedplasmid preparations of Em^(R) B. sphaericus P-1, transformed by pIL253(lanes 1-4), separated by agarose gel electrophoresis (ethidium bromidestained). Left panel. Autoradiogram of the hybridization between ³²P-labelled pIL253 and a Southern blot of the gel in the cental panel. Inall transformants, fragments specific for the introduced pIL253 plasmid(3.9 kb and 0.9 kb) are revealed. Right panel. Autoradiogram of thehybridization between the same blot as in the left panel, and ³²P-labelled pGVP1. Specific fragments (2.3-0.5 kb) from the endogenouspGVP1 are revealed.

FIG. 3 demonstrates the electrocompetence in B. sphaericus P-1 inlate-stationary phase. B. sphaericus P-1 cells were incubated at 37° C.in 100 ml LB broth on a gyratory shaker for 48 hr. Every 6 hr a samplewas withdrawn from the culture and colony-forming units/ml weredetermined. Cells were pelleted by centrifugation, washed andresuspended in 1 ml of distilled H₂). After addition of pIL253 DNA,cells were electroporated in 0.2 cm gapped electrocuvettes (E₀ 12 kV/cm,R=200 Ω), diluted in 900 μl LB, incubated for 1 hr at 37° C. and platedon LB plates with erythromycin.

FIG. 4 is a schematic representation of the high-copy number (pSL40) (A)and low-copy number (pSL84) (B) bifunctional vectors for E. coli and B.sphaericus spp. Restriction sites are indicated with their relativeposition on the physical map. Abbreviations used: bla: β-lactamase; MLS:resistance to the macrolide-lincosamide-streptogramin B group ofantibiotics; Orf E to G: open reading frames involved in replication inGram-positive hosts. pSL40 was constructed by ligating the 2.6-kbEcoRI/Xbal fragment of pLK68 in pIL253 (EcoRI/XbaI-linearized). Afterrestriction and filling- in at the EcoRI site of the resulting plasmid,the small multicloning site was exchanged for the polylinker of pJB66 bysubstituting the respective XbaI/BglII fragments. pSL84 was constructedby substituting the 1.8-kb Pstl/SalI fragment of pSL40 for the 1.3-kbPstI/SalI fragment of pACYC177 (containing the low copy number origin ofreplication for E. coli). In the resulting plasmid the 2.3-kb NsilI/XbaIfragment was replaced by the corresponding NsiI/XbaI fragment of pIL252containing the low-copy number origin of replication for Bacillus spp.

FIG. 5 is a restriction map of the genomic region containing the geneencoding the S-layer protein of B. sphaericus P-1. The black barrepresents the signal peptide-encoding sequence; the hatched bar showsthe mature part of SLP. The inserts of the four overlapping subclonesused for sequence analysis are depicted below the restriction map byopen bars. The arrow indicates the direction of transcription.

FIG. 6 shows the DNA sequence (SEQ ID NO:7) and deduced amino acidsequence (SEQ ID NO:8) of the slp gene of B.sphaericus P-1. The completeamino acid sequence (SEQ ID NO:9) is deduced. The putativeribosome-binding site preceding the SLP ORF is double underlined. Theshaded residues represent the signal peptide. The nucleotide sequence ofthe signal peptide is SEQ ID NO: 10, and its translation into aminoacids is SEQ ID NO: 11. The amino acid sequence (SEQ ID NO: 12) isdeduced. The mature SLP thus commences with amino acid residue 31. Thenucleotide sequence of the mature S-layer protein of Bacillus sphaericusP-I is SEQ ID NO: 13, and its translation into amino acids is SEQ ID NO:14. The amino acid sequence of the S-layer protein (SEQ ID NO: 15) isdeduced. Potential N-linked glycosylation sites are underlined. The stemof the Rho-independent transcription termination signal after thetranslation stop codon is indicated by arrows. The NH₂ -terminal aminoacid sequence determined by automated microsequence analysis of thepurified mature SLP is indicated by a dotted line. The nucleotidesequence of the NH₂ -terminal sequence is SEQ ID NO: 16, and itstranslation into amino acids is SEQ ID NO: 17. The amino acid sequenceis SEQ ID NO:18.

FIG. 7 shows the hydropathic profile of the B. sphaericus P-1 S-layerprotein by the computerized method of Kyte and Doolittle (1982).Horizontal bars represent potential transmembrane helices, as predictedby the method of Rao and Argos (1986).

FIG. 8 shows the sequence of the NH₂ -terminal portion of the SLP of B.sphaericus P-1 and the larvicidal strain 2362. The signal peptide (SEQID NO: 12) sequence of both proteins is boxed. Adjustments (horizontalbars) were introduced for optimal alignment. The amino acid sequence,which is shown in FIG. 8, of the SLP portion of B. sphaericus P-1 is SEQID NO: 19. The amino acid sequence, which is shown in FIG. 8, of the SLPportion of the larvicidal strain 2362 is SEQ ID NO:20.

FIG. 9:

A. Northern blot analysis of the slp-encoded mRNA in B. sphaericus P-1.

Total cellular RNA was isolated from different growth phases (see text).The internal 1.81 kb HpaI fragment of the slp gene was used for thegeneration of a ³² P-labelled probe. Migration pattern of molecular massmarkers as indicated.

B. Primer extension analysis of the transcriptional initiation sites ofthe slp-encoded transcripts. Two different primers were used (see FIG.10 and text for more details).

FIG. 10 shows the DNA sequence (SEQ ID NO:21) of the promoter regioncontrolling slp expression. The position of the 5' ends of thetranscripts, as determined by primer extension analysis, are indicated(black inverted triangles). The putative ribosome-binding site,preceding the slp ORF (shaded sequence) (SEQ ID NO:22) is indicated bydots. Primers used for primer extension assays were complementary to theoverlined sequences. The exact end points of the different deletionmutants (pSL151 to pSL159) are shown by an arrow. Potential -10 and -35boxes preceding the transcription initiation sites are indicated. Theputative -10 and -35 regions as reported by Bowditch et al. (1989) aremarked by an asterisk.

FIG. 11 shows how β-glucuronidase activity is directed by the differentslp promoter deletion mutants in B. sphaericus P-1 (hatched bars). InpSL87 the uidA gene is under control of the 138.sub..0. promoter.

FIG. 12 shows the effect of Ca²⁺ cations on the slp promoter-directedβ-glucuronidase activity in different deletion mutants. Black barsrepresent B. sphaericus P-1 cells grown in LB medium. Hatched barsindicate cells grown upon addition of 7 mM CaCl₂. Activity was measured4 hours after addition of CaCl₂ to the culture.

FIG. 13 shows the general outline of the strategy for disruptive singlehomologous recombination. AB₁ ^(R) : antibiotic resistance marker 1(Em^(R));AB₂ ^(R) : promoterless antibiotic resistance gene 2 (nptII);ori: origin of replication; wavy line: RNA transcript; P: promoter.

FIG. 14:

A. Restriction map of the bifunctional plasmid pSL64 used as based forthe construction of different intermediate vectors. Restriction sitesare indicated with their relative position on the physical map.Abbreviations used: bla: β-lactamase; MLS: resistance to themacrolide-lincosamide-streptogramin B group of antibiotics; OrfE to G:open reading frames involved in replication in Gram-positive hosts(Swinfield et al., 1990).

B. Nucleotide sequences upstream from the nptII-coding region (boxed) ofpSL64 (SEQ ID NO:23) and pSL101 (SEQ ID NO:24).

FIG. 15:

A. Schematic representation of carboxy-terminal truncated SLPs obtainedby single homologous recombination of the different intermediatevectors. Central block represents the restriction map of the chromosomalregion containing the slp gene. Hi: HindIII; Hp: HpaI; Bg: BglII; Pv:PvuII; Xb: XbaI. Black arrows represent SLPs in the wild-type andrecombinant P-1 strains as indicated on the left. Calculated molecularmasses of the SLPs are indicated on the right. Striped bars indicate theused subclones of slp gene. White bars indicate internal slp fragments,cloned in pSL64 in the different intermediate vectors.

B. SDS-PAGE of proteins from wide-type P-1 (lanes A), recombinantstrains P-1::pSL66 (lanes B), P-1::pSL68 (lanes C) and P-1::pSL69 (lanesD). lane M: high-molecular mass markers (Bio-Rad). Proteins are eitherTCA-precipitated from the supernatant of the cultures or are obtained bysonication and centrifugation. The insoluble fraction is indicated bydebris, whereas the soluble fraction is indicated as sonicate.

FIG. 16 is an autoradiogram of the hybridization between ³² P-labelledpSL20 and BglII-digested total DNA of P-1 (lane A), P-1::pSL69 (lane B),and P-1:: pSL102 (lane C). In P-1:: pSL69 and P-1::pSL102, the 1600 BPBglII fragment, hybridizing with P-1 total DNA has disappeared, whereastwo predicted fragments of 2600 and 6500 bp appeared.

FIG. 17 is a schematic representation of the peptides translationallyfused to carboxy-terminally truncated SLPs in the strains P-1::pSL102,P-1::pSL113 and P-1::pSL111. Central block represents the restrictionmap of the chromosomal region containing the slp gene, Hi: HindIII; Hp:HpaI; Bg: BglII; Pv: PvuII; Xb: XbaI. Arrows under the restriction maprepresent recombinant SLPs after integration of intermediate vectorsindicated above the restriction map. Black fragments represent SLPportion, striped bars indicate the S1 subunit of pertussis toxin, andwhite bars represent NPTII.

FIG. 18:

A. SDS-PAGE of total protein extract from P-1::pSL113 (lane 1),P-1::pSL102 (lane 2), and P-1::pSL69 (lane 3). Respective SLPs areindicated by a (130 kDa), b (102 kDa) and c (74 kDa).

B. Immunodetection on a Western blot of the gel in panel A, usinganti-NPTII antibodies. Two recombinant SLPs (indicated a and b) arerevealed.

C. SDS-PAGE of total protein extract from P-1::pSL111 (lanes 1-3),P-1::pSL102 (lane 4) and P-1::pSL69 (lane 5).

D. Immunodetection on a Western blot of the gel in panel C, usinganti-PT antibodies. Two recombinant SLPs (of 90 and 120 kDa) arerevealed (d* and d, respectively).

FIG. 19 is an autoradiogram of in gel kanamycin phosphorylation assay onprotein extracts separated by non-denaturing polyacrylamide gelelectrophoresis extracted from P-1 (panel A) P-1::PSL69 (panel B), andP-1::pSL102 (panel C). Significant phosphorylating activity in thehigh-molecular mass region can only be observed in P-1::pSL102.

FIG. 20 shows immunogold labelling on intact bacteria using anti-NPTIantibodies. Panel A, P-1::pSL69, panel B, P-S::pSL102; panel C,P-2::pSL113. Significant accumulation of gold-label can only be observedin P-a::pSL102 and to a lesser extent in P-1::pSL113.

FIG. 21 shows the detection of PT subunit S1 and NPTII in native sacculiprepared from P-1::PSL102 and P-1::pSL111. CE: cellular extracts; NS:native sacculi.

EXAMPLE 1

1. Materials and Methods

Bacterial strains and plasmids. In Table I, the bacterial strains andplasmids used in this study are listed. B. sphaericus strains were grownin Luria-Bertani (LB) broth (Miller, 1972), supplemented with 0.7% agarfor solid media. Selective antibiotic concentrations for B. sphaericuswere: 10 μg/ml erythromycin (Em); 10 μg/ml nalidixic acid (Na). For E.coli, 200 μg/ml of triacillin was used.

                  TABLE I    ______________________________________    Bacterial strains and plasmids used in this study            Characteristics   References    ______________________________________    E. coli    DH5α              F.sup.-, .o slashed.80dlacZΔM15,                                  Hanahan              Δ(lacZYA-argF).sub.U169, recA1,                                  (1983)              endA1, hsdR17(r.sub.k.sup.-, m.sub.k.sup.+), supE44    MC1061    hsdR, hsdM, hsdS, araD139,                                  Casadaban              Δ(ara--leu).sub.7697, Δlac.sub.x74,                                  and Cohen              galK, rpsL          (1980)    B. sphaericus    P-1       Nalidixic acid resistant                                  Lewis et al.                                  (1987)    1593                          BGSC    10208                         ATCC    Lactococcus latis    MG1363                        Gasson and                                  Davies (1980)    Plasmids    pGVP1     natural isolate in B. sphaericus P-1                                  This study    pGVP2     cointegrate on BamHI-linearized                                  This study              pUC9 and Bg1II-linearized pGVP1    pUC9      Ap.sup.R            Vieira and                                  Messing                                  (1982)    pPGV5     bifunctional, Ap.sup.R, Nm.sup.R                                  This study    pJB66     Ap.sup.R            Botterman                                  and Zabeau                                  (1986)    pSL40     cointegrate of pJB66 and pIL253,                                  This study              Ap.sup.R, MLS.sup.R    pSL84     cointegrate of pACYC177 and                                  This study              pIL252, Ap.sup.R, MLS.sup.R    pAMβ1              MLS.sup.R, autotransmissible, natural                                  Clewell et.              isolate             al (1974)    pIL252    MLS.sup.R, low-copy number vector                                  Simon and              derived from pAMβ1                                  Chopin                                  (1988)    pIL253    MLS.sup.R, high-copy number vector                                  Simon and              derived from pAMβ1                                  Chopin                                  (1988)    pACYC177  Ap.sup.R, Km.sup.R, low-copy number vector                                  Chang and                                  Cohen (1978)    ______________________________________

Transformation and plasmids. Competent E. coli strains were prepared andtransformed according to Kushner (1978). Transformation of B. sphaericusP-1 intact cells was achieved by electroporation using the protocoldeveloped as described in the Results section below. B. sphaericus P-1cells, grown in LB broth for 42 hr at 37° C. on a gyratory shaker, wereharvested by centrifugation (9000 g), washed with ice-cold distilled H₂O, resuspended in 1/10 volume of a 10% glycerol solution in distilledH₂), aliquoted in 100 μl samples, and stored at -70° C.

For transformation, samples were quickly thawed, mixed with DNA, andtransferred into 0.1 cm gapped electrocuvettes. An electrical pulse (14kV/cm, 25 μF) was delivered, using a GenePulser (Trade Mark) apparatus(Bio-Rad laboratories) with Pulse Controller extension set at 200 Ω.After the electrical pulse was delivered, cells were diluted with 900 μlof LB broth and incubated at 37° C. for 1 hr, prior to plating on solidLB medium, and supplemented with appropriate antibiotics.

General recombinant DNA techniques. E. coli plasmid DNA was preparedaccording to Sambrook et al. (1989), whereas for plasmid and total DNApreparations of B. sphaericus, cells were pretreated with lysozyme (100μg/ml) at 37° C. for 10 min. Restriction enzymes were purchased from NewEngland Biolabs, Pharmacia (Uppsala, Sweden) or Bethesda ResearchLaboratories, and were used according to the manufacturers'recommendations.

Elution of DNA restriction fragments was done using GeneClean II (TradeMark) kit (Bio101 Inc., La Jolla, Calif., US). Filling-in of protrudingsingle-stranded termini after restriction enzyme digestion (using Klenowor T₄ DNA polymerase) and ligations were done according to standardconditions (Sambrook et al., 1989). Southern transfer and hybridizationwere performed using Hybond N⁺ membranes (Amersham) and QuickPrime(Trade Mark) labelling kit (Pharmacia) to prepare ³² P-labelled probes,except for blotting of single-stranded DNA, which was achieved usingnitrocellulose membranes.

2. Results

Characterization of endogenous plasmids of B. sphaericus P-1. Plasmidpreparations, according to the alkaline lysis method, followed byequilibrium density gradient centrifugation, revealed the presence of asmall plasmid (2.8 kb), designated pGVPI, in B. sphaericus P-1. Bypreliminary restriction analysis of pGVPI, a unique restriction site forBglII was found. For further restriction enzyme analysis, a cointegrateplasmid (pGVP2) was constructed, by joining BglII-linearized pGVP1 andBamHI-linearized pUC9, allowing large-scale preparations from E. coli.Single- and double-occurring restriction enzyme sites (BglII, AvaI,NcoI, PstI, HindIII, SspI) were ordered by appropriate double digestions(FIG. 1A). No sites were founds for KpnI, BamHI, EcoRI, ApaI, ClaI,EcoRV and SphI.

Hybridizations were performed between ³² P-labelled pGVPI and Southerntransfers of non-denatured total DNA of B. sphaericus P-1 tonitrocellulose membranes (a frequently used method for detection ofsingle-stranded replication intermediates in Gram-positive replicons;Gruss and Ehrlich, 1989). A specific hybridization signal was observedin undigested total DNA (FIG. 1B, panel 2, lane A), corresponding to asingle-stranded intermediate. The signal was not detected in DNA sampleswhich were digested either with S1 nuclease or with the single-strandedDNA-cleaving endonuclease HhaI, prior to gel separation.

Treatment with T₄ polymerase, which degrades specific linearsingle-stranded DNA, did not decrease the signal, indicating that thepPGV1 replication intermediate is circular. As it is of considerableinterest to use B. sphaericus P-1 as a host for transformationexperiments several methods for plasmid curing that proved successful inGram-positive bacteria including novobiocin (Gonzales et al., 1981),rifampin (Johnston and Richmond, 1970), sodium dodecyl sulfate (Sonsteinand Baldwin, 1972)! were tried out but did not result in the productionof a plasmid-free strain.

Introduction of pAMβ1 into B. sphaericus P-1 by intergenericconjugation. To test whether the macrolide lincosamide steptogramin B(MLS) resistance determinant and the origin of replication of theauto-transmissible plasmid pAMβ1 (26.5 kb) were functional in B.sphaericus P-1, this plasmid was introduced into P-1 by conjugating itwith Lactococcus lactis MG1363 pAMβ1! (Gasson and Davies, 1980). Thisplasmid was chosen because it had previously been introducedsuccessfully into B. sphaericus 1593 (Orzech and Burke, 1984). Afterovernight incubation of a mixture of both strains (ratio 1:1) onnitrocellulose filters placed on M17⁺ lactose medium at 37° C., bacteriawere collected and several dilutions were plated on LB mediumsupplemented with Em.

Em-resistant B. sphaericus P-1 colonies were obtained at a frequency of3×10⁶ (transconjugants/acceptor strain). After colony purification on LBmedium supplemented with erythromycin and nalidixic acid, putativetransconjugants were analyzed for the presence of pAMβ1 by Southernhybridization using ³² P-labelled pAMβ1 as a probe (data now shown). Theplasmid was stable for several generations, even in the absence ofselective pressure. These results prompted us to use pAMβ1-derivedcloning vectors (e.g. pIL253) for electrotransformation experiments inP-1.

Electrotransformation. Initial experiments using pIL253 to transform B.sphaericus strains, following reported protocols forelectrotransformation of several Bacilli (Takagi et al., 1989; Bone andEllar, 1989; Taylor and Burke, 1990) were unsuccessful. The commondenominator in these protocols is the use of cells harvested in early ormid-log growth phase. However, using cells harvested from late-logsolid-grown colonies, which were washed once with ice-cold distilled H₂O, and standard electrical parameters for E. coli (12 kV/cm, 200 Ω, 25μF in 0.2 cm gapped electrocurvettes), 10² transformants were obtained.Plasmid analysis revealed the presence of two plasmids that could beidentified as the endogenous pGVP1 and the introduced pIL253 by Southernhybridization (FIG. 2).

To optimize the physiological conditions for electroporation of P-1,cells harvested at different time-points in a growing culture werewashed once and resuspended in 1/10 volume distilled H₂ O, andelectroporated (at 12 kV/cm, 200 Ω, 25 μF in 0.2-cm gappedelectrocuvettes). Although growth of the culture stagnated after 8 hr,transformants were not obtained until 36 hr of incubation. The number oftransformants reached a maximum at 42 hr incubation, and significantlydecreased after 48 hr of incubation, presumably due to cell death (FIG.3). This phenomenon demonstrates the need for a certain physiologicalstate of the bacterial cells required for electrotransforrnation, or inother words electrocompetence. Electrocompetence has been inferred toexplain saturation of transformation efficiencies at increasing DNAconcentration (Chassy et al., 1988; Desomer et al., 1990).

Addition of different chemicals (used to increase transformationefficiency in protocols for different bacterial species) to theelectroporation medium, such as polyethylene glycol (PEG) 1000 (15% w/v)and glycerol (10% w/v) improved the transformation efficiencysignificantly. (Table II). Variation of the electrical parametersincluded transformation at higher voltages (12, 14 and 16 kV/cm in0.1-cm gapped electrocuvettes) and use of different external resistances(200, 400 and 600 Ω). Maximum transformation efficiencies were obtainedat 14 kV/cm, 25 μF, 200 Ω using 0.1-cm gapped electrocuvettes (Table IIbelow).

Combination of both improved protocols, as described in the Materialsand Methods section above, routinely yielded 10⁵ transformants per μgDNA. Cells could be kept frozen at -70° C. without significant loss ofelectrocompetence.

The high transformation efficiency obtained by this protocol prompted usto test whether plasmids with single-stranded replication intermediates(such as the pUB110-derived vector pPGV5) could be used as transformingDNA, and eventually yield a P-1 strain, cured of pGVP1 byincompatibility. pPGV5 is a cointegrate via the EcoRI site of pUC4(Vieira and Messing, 1982) and pPL703 (Mongkolsuk et al, 1983).Nm-resistant transformants were obtained with low frequency (Table IIbelow) and contained intact pPGV5 in addition to the endogenous pGVP1(data not shown).

Application of the same protocol to B. sphaericus 1593 and ATCC 10208yielded no transformants (Table II below). Indeed, a previouslypublished protocol for electrotransformation for B. sphaericus 1593 usedcells harvested in early-log growth-phase (Taylor and Burke, 1990).

Construction of bifunctional vectors for E. coli and B. sphaericus.Bifunctional vectors that can replicate in both B. sphaericus and E.coli have the advantage that cloning procedures and analysis can be donewith well established methods in E. coli prior to introduction of thefinal construct into B. sphaericus. Bifunctional plasmids wereconstructed with high- (PSIA40) and low-copy number (pSL84) in bothhosts. pSL40 contains the multilinker, ampicillin-resistance gene andthe Co1E1 origin of replication of pJB66 (Botterman and Zabeau, 1980) aswell as the MLS determinant and origin of replication of pIL253 (FIG.4A). In pSL84, the high-copy number origin of replication of pJB66 isexchanged for the low-copy number origin of pACYC177, whereas the originof pIL253 is replaced for that of pIL252, an ancestral plasmid of pIL253with low-copy number (FIG. 4B). Upon introduction in B. sphaericus P-1,both plasmids exhibited the expected copy number control. No significantdifference in transformation efficiency was observed using pSL40 orpSL84 prepared from either the E. coli MC1061 or the B. sphaericus P-1hosts (Table II below).

                  TABLE II    ______________________________________    Transformation efficiencies using different electrical    conditions, B. sphaericus strains and plasmids           Electroporation                      E.sub.o R           Transformation    Strain medium     kV/cm   Ω                                    Plasmid                                          efficiency.sup.d    ______________________________________    P-1    H.sub.2 O   0       200p IL253 <10.sup.1    P-1    H.sub.2 O   6      200   pIL253                                          2.0 × 10.sup.1    P-1    H.sub.2 O  10      200   pIL253                                          3.4 × 10.sup.2    P-1    H.sub.2 O  12      200   pIL253                                          8.5 × 10.sup.2    P-1    H.sub.2 O   12.sup.a                              200   pIL253                                          9.6 × 10.sup.3    P-1    H.sub.2 O   14.sup.a                              200   pIL253                                          6.7 × 10.sup.3    P-1    H.sub.2 O   16.sup.a                              200   pIL253                                          8.6 × 10.sup.2    P-1    H.sub.2 O   14.sup.a                              400   pIL253                                          5.3 × 10.sup.3    P-1    H.sub.2 O   14.sup.a                              600   pIL253                                          4.7 × 10.sup.2    P-1    30% PEG 1000                      12      200   pIL253                                          <10.sup.1    P-1    15% PEG 6000                      12      200   pIL253                                          4.0 × 10.sup.1    P-1    15% PEG 1000                      12      200   pIL253                                          2.4 × 10.sup.2    P-1    10% glycerol                      12      200   pIL253                                          1.4 × 10.sup.3    P-1    10% glycerol                       14.sup.a                              200   pIL253                                          6.5 × 10.sup.5    P-1    H.sub.2 O  12      200   pPGV5.sup.b                                          .sup.  8.0 × 10.sup.1c    1593   H.sub.2 O  12      200   pIL253                                          <10.sup.1    10208  H.sub.2 O  12      200   pIL253                                          <10.sup.1    P-1    10% glycerol                       14.sup.a                              200   pSL40 2.0 × 10.sup.3    P-1    10% glycerol                       14.sup.a                              200   pSL40.sup.b                                          2.6 × 10.sup.3    ______________________________________     .sup.a in 0.1 cm gapped electrocuvettes;     .sup.b plasmid source was E. coli;     .sup.c selected on LB with Nm;     .sup.d number of transformants selected on LB EM per μg of DNA.

Example 2

1. Materials and Methods

Bacterial strains, plasmids and media. Bacterial strains used are B.sphaericus P-1 (Lewis et al, 1987) and B. subtilis BR151 BacillusGenetic 10 Stock Center, Ohio State University, Columbia). E. coli hostswere either DH5α (Hanahan, 1983) or MC1061 (Casadaban and Cohen, 1980).Cloning was performed in pUC18 (Yanisch-Perron et al, 1985). pSL40(Example 1) is a high copy number E. coli-Bacillus shuttle vector,essentially composed of pJB66 (Botterman and Zabeau, 1987) and pIL253(Simon and Chopin, 1988). The β-glucuronidase (uidA) gene cassette wasisolated from pGUSl (Peleman et al, 1989) and introduced into pSL40 as aBamHI/SphI fragment, yielding pSL150. Bacteria were grown on LB medium(Miller, 1972) solidified with 1.5% agar, whereas liquid cultures weregrown in TB medium (Tartof and Hobbs, 1987). When required antibioticswere added: ampicillin (100 μg/ml) or erythromycin (10 μg/ml). Allcultivations were performed at 37° C.

DNA techniques. Recombinant DNA techniques for E. coli were performedaccording to standard conditions (Sambrook et al, 1989). Restriction andmodifying enzymes were purchased from Pharmacia, New England Biolabs,Promega or Bethesda Research Laboratories and used according to theirrecommendations. DNA fragments were purified from agarose gel using theGene Clean Kit (Bio-101 Inc.). DNA sequences from both strands weredetermined by the dideoxy-chain termination method (Sanger et al, 1977)using the T7 Sequencing Kit (Pharmacia). Sequence analysis was carriedout with the Intelligenetics suite of program (Intelligenetics Inc.).Databases were screened by FASTDB software (Brutlag et al, 1990).Unidirectional deletions were generated by combined ExoIII/S1 nucleaseactivity, using the Double-stranded Nested Deletion Kit (Pharmacia).Oligonucleotides were synthesized on an ABI 394 DNAIRNA Synthesizer(Applied Biosystems Inc.). High voltage transformation of E. coli DH5αwith ligation mixtures was done with a Bio-Rad Gene Pulser (Trade Mark).Site-specific mutagenesis using polymerase chain reaction (PCR) wasperformed as described (Landt et al, 1990).

Construction and screening of libraries. B. sphaericus P-1 genomic DNAwas prepared as described by Mielenz (1983) and digested to completionwith the appropriate restriction enzyme. Libraries were constructed indigested and dephosphorylated pUC18, according to standard conditions(Sambrook et al, 1989). 3840 colonies were transferred to Hybond-N nylonmembranes (Amersham International) and screened by colony hybridizationunder standard stringency conditions. ³² P-labelled probes weregenerated using the ^(T7) Quick Prime (Trade Mark) Kit (Pharmacia).

RNA analysis. Total RNA was extracted from B. sphaericus P-1 by thehot-phenol method of Aiba et al (1981). Total RNA isolated at differentgrowth phases was run on a formaldehyde containing agarose gel andtransferred to Hybond-N nylon membranes (Pharmacia) and hybridizedaccording to the manufacturer's recommendations. Single strandedoligonucleotides (FIG. 10) were used as primers in a primer extensionassay. 50 μg of RNA was mixed with 50 ng primer andethanol-precipitated. The pellet was resuspended in 20 μl5×hybridization buffer (2 M NaCl, 50 mM PIPES pH 6.4, 5 mM EDTA) and 80μl deionized formamide and incubated for 15 minutes at 85° C. Primerannealing proceeded over-night at 37° C. Primed RNA wasethanol-precipitated. The extension reaction was carried out for 90minutes at 42° C. in a 40 μl mixture containing 50 mM Tris.HCl (pH 8.2),10 mM dithiothreitol, 6 mM MgCl₂, 25 μg/ml actinomycin D, 250 μM dCTP,dGTP, dTTP, 150 μM dATP, 60 μCi α-³⁵ S! dATP (Amersham International)and 40 units Reverse Transcriptase. Upon completion of the reaction 2 μlpancreatic RNase (1 mg/ml) was added and incubated for another 20minutes. Extension products were purified by phenol/chloroformextraction and subsequent ethanol precipitation. The size of theextended products was deduced by comparison to a corresponding sequenceladder, generated with the same primer.

Protein micro-sequence analysis. The surface-layer protein from B.sphaericus P-1 was isolated from cell walls by urea extraction asdescribed by Lewis et al (1987). Upon sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) proteins wereelectro-blotted and immobilized on a treated glass fibre plate (Bauw etal, 1987) for NH2-terminal amino acid sequence determination on anautomated ABI 473A Protein Sequencer (Applied Biosystems Inc.).

Enzyme assay. β-glucuronidase activity was measured essentially asdescribed (Jefferson et al, 1986) using p-nitrophenyl-β-D-glucuronide assubstrate. Cells were resuspended in 1 ml reaction buffer, supplementedwith 0.01% SDS. Permeabilization of bacterial cells was achieved byaddition of 25 μl chloroform and vortexing for 10 seconds. Aftertermination of the reaction, cells were pelleted and the clearedsupernatant was used for O.D. measurement.

2. Results

Identification and cloning of the sip gene. The surface-layer protein ofB. sphaericus P-1 was purified and subjected to automated microsequenceanalysis. 21 NH₂ -terminal amino acid residues (SEQ ID NO: 18) could bededuced: NH₂-Ala-Gln-Val-Asn-Asp-Tyr-Asn-Lys-Ile-Ser-Gly-Tyr-Ala-Lys-Glu-Ala-Val-Gln-Ala-Leu-Val.Based on residues 11 to 19 of this sequence, a specificoligodeoxynucleotide probe mixture was synthesized with the followingsequence (SEQ ID NO:25): 5'-GCYTGIACIGCYTCYTTIGCITAICC-3' wherein Y is Cor T, and wherein I is inosine. Several unique bands were revealed onSouthern blots of restricted genomic DNA when hybridized to the ³²P-labelled oligonucleotide probe.

A preliminary restriction map was established from which it was deducedthat the 5.0-kb EcoRI fragment very probably contained the complete slpgene. An EcoRI-generated library of B. sphaericus P-1 genomic DNA inpUC18 was screened by colony hybridization. Despite the use of differenthybridization conditions no subclones containing the 5.0-kb fragmentcould be isolated, suggesting that cloning of the entire gene or a largepart of it in E. coli is lethal to the host. Therefore cloning ofsmaller restriction fragments, identified in the Southern blot analysis,was pursued.

Screening of a HindIII-generated library resulted in the isolation of apUC18 clone (pSL1), containing a 1.8-kb insert. Further analysis showedthat the homology could be delineated to a small 100-bp HindIII/PvuIIfragment. Sequence analysis confirmed this homology: the deduced aminoacid sequence of this region completely matched the sequence as obtainedafter microsequencing of the SLP subunit. Preceding this region atypical signal peptide sequence for secretion was detected. The pSLlclone thus contains the slp promoter and a stretch encoding a 30-residuesignal peptide and the first 20 amino acid residues of the SLP of B.sphaericus P-1.

It also became clear that the originally identified 5.0-kb EcoRifragment indeed contained the complete slp gene, including its ownpromoter. However due to inability to clone this fragment in E. coli, wewere obliged to isolate the gene as a set of overlapping clones. In thisway, three other pUC18 clones were isolated from several libraries usingthe previous fragment as probe: pSL4, containing a 0.8-kb PvuIIfragment; pSL10, harbouring a 1.6-kb BglII fragment and pSL20 carrying a3.0-kb HindIII fragment (FIG. 5). In total a region of 4.6 kb wasspanned from which the DNA sequence was determined (FIG. 6).

The slp gene sequence. Analysis of the DNA sequence starting from theEcoRI site to the most downstream HindIII site revealed an open readingframe (ORF) of 3756 nucleotides, starting at the ATG initiation codon(position 95 to 97; FIG. 6) and terminating at the stop codon TGA(position 3851 to 3853; FIG. 6), whereas as many as 256 translation stopcodons are dispersed over the 2 other reading frames. This ORF couldencode a polypeptide of 1252 residues with a deduced molecular mass of130,060 Da, which is 20 kDa less than the value deduced from SDS-PAGE.This discrepancy is probably due to the fact that the B. sphaericus P-1SLP is glycosylated (Lewis et al, 1987). Indeed, 20 potential N-linkedglycosylation sites are distributed over the sequence. The protein has acalculated pI value of 4.59, which is in accordance with theexperimentally determined value of 4.6±0.4 (Lewis et al, 1987).

The 3' end of the ORF is followed by a palindrome with a stem of 13 basepairs (position 3904 to 3933; FIG. 6) and a thymidine-rich stretch,which is typical for a Rho-independent transcription termination signal(Platt, 1986). The start codon ATG is preceded by a potentialribosome-binding site 5'-AGGGAGG-3' (position 78 to 85; FIG. 6). The 11nucleotide-spacing between the middle A of this motif and the ATG codonis typical of that found in gram-positive bacteria (Hager andRabinowitz, 1985).

The deduced amino acid sequence was analyzed by the computerized methodof Kyte and Doolittle (1982) for hydropathicity (FIG. 7). The NH₂-terminal sequence (30 residues) appears to be very hydrophobic, whichis in accordance with the presence of a signal peptide responsible forsecretion. It moreover ends by the sequence VASA, a motif frequentlyrecognised by signal peptidases (von Heijne, 1986) and is then directlyfollowed by the sequence determined by microsequence analysis of themature SLP subunit. Several other hydrophobic regions, which mightinterfere with membrane translocation were observed at the COOH-terminus(FIG. 7).

Table III shows the amino acid composition of the SLP, which sharesseveral features with other S-layer proteins. The onlysulphur-containing amino acid is methionine (2 residues). It contains ahigh proportion of hydrophobic amino acids (38%), but it is not veryenriched in acidic residues (10.6%) versus basic residues (8.3%), as isthe case for the two S- layer proteins of B. brevis 47 (Tsuboi et al,1988). No significant homology to other S-layer proteins was found,except with the SLP of B. sphaericus 2362 (Bowditch et al, 1989), at thelevel of the NH₂ -terminal sequence (FIG. 8). The first 200 residuesshow a degree of 82% identity. However, no homology downstream of thisregion could be detected. This observation was confirmed by comparingthe hydropathicity plots of both proteins (data not shown).

                  TABLE III    ______________________________________    Amino acid composition of the B. sphaericus P-1 SLP    Amino Acid     Number  % (molecular mass)    ______________________________________    Threonine      166     12.8    Valine         134     10.2    Alanine        181     9.9    Lysine         91      9.0    Asparagine     88      7.7    Glutamic acid  69      6.9    Leucine        64      5.6    Aspartic acid  62      5.5    Phenylalanine  47      5.3    Serine         78      5.2    Isoleucine     55      4.8    Tyrosine       37      4.6    Glycine        95      4.2    Glutamine      34      3.4    Proline        31      2.3    Arginine       11      1.3    Tryptophan      6      0.9    Methionine      2      0.2    Histidine       1      0.1    Cysteine        0      0.0    ______________________________________

The sip promoter. In order to examine how the slp gene is transcribed invivo total RNA was isolated at different growth phases: early and middlelogarithmic phase, early stationary phase and from an overnightsaturated culture. Northern blot analysis demonstrated the presence ofone single transcript of approximately 4500 nucleotides. The sip gene isexpressed at high level up to early stationary phase. However a sharpdecrease is observed in a saturated culture, together with asimultaneous drop in rRNA levels due to stringent response (Cashel andRudd, 1987) (FIG. 9). These high levels of expression during most of thebacterial growth cycle are to be expected in view of the continuous needof large amounts of SLP subunits for the assembly of an intactsurface-layer, even at stationary growth phase when the SLP is releasedinto the medium (Howard and Tipper, 1973).

Primer extension assays were performed to study the existence of complexmultiple promoters involved in regulation of slp gene expression, suchas in the case of B. brevis 47 (Adachi et al, 1989). Using two differentprimers the 5' end(s) of the transcript were detected. Three differenttranscription initiation sites were identified in both experiments atpositions -184 (P1), -340 (P2) and -385 (P3) with respect to the firstnucleotide (+1) of the start codon (FIG. 10). Each transcription startsite was preceded by a potential -10 and -35 motif as indicated in FIG.10. Spacing between both motifs corresponded to the preferred internallength (16 to 18 bp) for B. subtilis promoters (Moran et al, 1982).

β-Glucuronidase fusions to study sip gene expression. The slp promoterwas fused to the β-glucuronidase (uidA) reporter gene to examine theexpression characteristics of this complex 5'-upstream region. ThroughPCR-mediated site-specific mutagenesis a Ncol site was generated at theATG start codon of the slp gene. The promoter was then isolated as aXbaI/NcoI fragment and fused to the uidA ORF at the NcoI site in pSL150,yielding pSL151.

Through the combined action of ExoIII/S1 nuclease a set of progressivedeletions towards the ATG start codon was generated and introduced intopSL150 as XbaI/NcoI fragments. The exact end point of each deletion wasdetermined by sequence analysis (FIG. 11). This set of plasmids (pSL151to pSL159) was introduced into B. sphaericus P-1 byelectrotransformation as described in Example 1 and β-glucuronidaseactivity was monitored.

The results are shown in FIG. 11 and can be summarized as follows:deletions up to approximately position -150 are completely abolished inuidA expression. Indeed, according to the primer extension assay thesemutants are devoid of any of the three identified promoters. Deletionsremoving sequences up to position -375 show a threefold increase inβ-glucuronidase activity as compared to pSL151. These constructs onlycontain promoter P1. All smaller deletions show again wild-type levelsof β-glucuronidase activity. In these mutants all three promoters areintact again.

Effect of Ca²⁺ on slp expression. In several cases it has been reportedthat Ca²⁺ plays a key role in the assembly of the surface-layer on thebacteria (Feraldo et al, 1991 Yang et al, 1992). Moreover, Adachi andco- workers (1991) observed that Ca²⁺ repressed the expression of thecell wall protein gene operon of B. brevis 47. In this context thepreviously constructed Pslp-uidA fusions proved to be excellent tools tomonitor the possible effect of Ca²⁺ on slp gene expression. Bacteriawere grown in LB medium supplemented with an overdose Ca²⁺ (7.5 mM) andcompared to cells grown in the absence of Ca²⁺. β-glucuronidase activitywas measured 4 hours after dilution of the cultures (1/100) andsimultaneous addition of Ca²⁺ to the medium. As can be seen in FIG. 12,addition of Ca²⁺ resulted in a two-fold reduction of β-glucuronidaseactivity in all mutants up to position -440, whereas mutants containingonly promoter P1 were immune to this negative effect. These resultssuggest that the Ca²⁺ repression is located at promoters P2 and/or P3.These observations were confirmed when assaying enzyme activity 24 hoursafter addition of Ca²⁺ (data now shown).

Example 3

1. Materials and Methods

Bacterial strains and plasmids. Growth media and selective antibioticconcentrations for B. sphaericus P-1 have been described in Example 1.The plasmid pSL64, used as a basis for the construction of the differentintermediate vectors, was isolated by insertion of a promoterless nptIIgene as a 1.12-kb BamHI/SalI fragment from pKm109/2 (Reiss et al, 1984a)into the bifunctional, erythromycin resistance (Em^(R)) encoding vectorpSL40 of Example 1 (FIG. 14A). The nucleotide sequence of the linkerpreceding the nptII gene in pSL64 is shown in FIG. 14B1.

A similar plasmid (pSL101) was constructed by exchange of the BamHI/NcoIfragment of pSL64 for a similar sized fragment of pLKM92 to shift thereading frame of the nptII gene, compared to the multicloning site.pLKM92 is pKm109/90 (Reiss et al, 1984a) with a slightly modifiedpolylinker. The nucleotide sequence of the linker preceding the nptIIgene in pSL101 is shown in FIG. 14B2.

pSIA, pSL10, and pSL20 are subclones of the slp gene of Example 2 andare indicated on FIG. 15. Intermediate vectors, constructed in thecourse of this study are summarized in Table IV:

                  TABLE IV    ______________________________________    Intermediate Vectors    Plasmid Name               Cloned internal part of SLP.sup.a                                Cloning Vector    ______________________________________    pSL66      2048-2854        pSL64    pSL68      1470-2476        pSL64    pSL69      443-2053         pSL64    pSL70      443-1609         pSL64    pSL71      49-807           pSL64     pSL102    443-2053         pSL101     pSL111    443-2053         .sup. pSL64.sup.b     pSL113    2084-2854         pSL101    ______________________________________     .sup.a bp 1 is the A base of the start codon;     .sup.b containing the 554bp Sau3A fragment encoding the central part of     S1.

Transformation. Competent E. coli MC1061 strains were prepared andtransformed according to Kushner (1978), whereas transformation of B.sphaericus P-1 was achieved by electroportation as described in Example1.

Sacculi preparation. Native sacculi were prepared by a protocoldescribed by Sara and Sleytr (1987) and modified for B. sphaericus P-1or derivative strains were grown overnight at 37° C. in TB medium(Tartof and Hobbs, 1987) on a gyratory shaker. Cells were harvested bycentrifugation, resuspended in 50 mM Tris-HCl, pH 7.2 (50 ml per 100 gpellet), and sonicated for 1 min (40 Watt, using a Bransic Sonic PowerCo. sonicator). Triton X-100 was added to a final concentration of 2%,and the mixture was incubated, with agitation, for 30 min at 50° C.Treated cells were collected by centrifugation (15,000 g, 10 min), andwashed three times with cold, distilled H₂ O. The pellet was resuspendedin 5 mM MgCl₂, containing DNAse (5 μg/ml) and RNAse (20 μg/ml), andincubated for 15 min at 37° C. The resulting native sacculi werepelleted, washed three times with cold distilled H₂ O, and resuspendedin 20 ml buffer (20 mM Tris-HCl, pH 7.2, 2.5 mM CaCl₂, and 2 mMphenylmethylsulfonylfluoride).

Enzymatic assays. Enzymatic assays were performed either ontrichloroacetic acid (TCA)-precipitated culture supernatants or oninsoluble and soluble fractions of sonicated cells (4 times 10 sec, 40W). NPTII activity was assayed by the in situ phosphorylation assayafter separation of the proteins on non-denaturing polyacrylamide gels(Reiss et al, 1984b). Nicotinamide adenine dinucleotide (NAD)glycohydrolase activity was measured as the release of ¹⁴ C-labellednicotinamide from carbonyl-¹⁴ C!NAD as described by Locht et al, (1987).

SDS-PAGE and immunoblotting. Sodium dodecyl sulfate polyacrylamide gelelectrophoresis and Western blotting were performed by standardprocedures (Laemmli, 1979). The filters were blocked with 2% Tween 20and incubated with an 1:1000 dilution of the specific rabbit (anti-NPTII) or goat (anti-pertussis toxin PT!) serum, followed by alkalinephosphatase conjugated goat anti-rabbit or mouse anti-goat IgG (Bio-Rad)as described by the manufacturers.

Immunogenicity of recombinant bacteria with composite S-layers in mice.Groups of five female Balb-C mice were injected (intraperitoneally) withdifferent titers of recombinant P-1::pSL111 bacteria (10⁷, 10⁸, and 10⁹colony forming units (CFU)), corresponding to an estimated amount of0.1, 1, and 10 μg of S1 subunit of pertussis toxin, either with orwithout Freund's adjuvant. Control experiments included purified S1subunit of PT, and recombinant P-1::pSL102 bacteria. Injections wererepeated after 3 and 9 weeks. At week 12, sera samples were collected.Mice sera were screened for the presence of antibodies directed againstPT S1 subunit in a sandwich ELISA. PT was used as antigen and capturedby a Guinea pig antiserum.

General recombinant DNA techniques. These were according to Sambrook etal, (1989). Restriction enzymes were purchased from New England Biolabs,Pharmacia or Bethesda Research Laboratories and were used according tothe manufacturers' recommendations. Elution of DNA fragments separatedby agarose gel electrophoresis was done using Gene Clean (Trade Mark)kit (Bio101 Inc, La Jolla, Calif., US). Southern transfer andhybridization were performed using Hybond N⁺ membranes (Amersham) andQuickprime (Trade Mark) labelling kit (Pharmacia) to prepare ³²P-labelled probes.

2. Results

Analysis of carboxy-terminal deletions of B. sphaericus P-1 SLP.Sequence analysis of the 4.5-kb slp gene of B. sphaericus P-1 revealedthat the predicted first 200 amino acids of SLP were highly similar tothe deduced sequence of the SLP of B. sphaericus 2362 (Bowditch et al,1989), whereas the remaining parts of the proteins were highlydivergent. Potential transmembrane helices were predicted to be locatednear the carboxy-terminal ends of both proteins (Bowditch et al, 1989).Therefore, these conserved motifs might be important in the build-up oranchoring of the S-layer. The fraction of the non-homologousCOOH-terminal part of B. sphaericus P-1 SLP that could be removedwithout notable interference with S-layer assembly was determined byprogressive deletions.

Therefore, several intermediate vectors were constructed (based on thebifunctional vector pSL64; Table IV) that contained different internalfragments of the slp gene, cloned upstream of a promoterless nptII gene(pSL66, pSL68, pSL69, pSL70, pSL71; Table IV and FIG. 15A). Theseconstructs were introduced into B. sphaericus P-1 by electroporation andselection for Em^(R) transformants. Neomycin-resistant (NmR) colonies,and thus putative single homologous recombinants, were obtained for theP-1 strains transformed by almost all intermediate vectors, except forpSL71 which contained the most amino-terminally located fragment of theslp gene.

Southern analysis revealed the patterns expected for single homologousrecombination (see FIG. 16 for pSL69 integration). Culture supernatants,insoluble cell debris, and soluble cell contents after sonication of thestrains carrying the different carboxy-terminal deletions were analyzedby SDS-PAGE electrophoresis (FIG. 15B). Abundant amounts of proteinswith the expected molecular masses (see FIG. 15A) could be readilyobserved in the insoluble cell fraction. In contrast with the ratherabundant presence of SLP in culture supernatants of wild-type P-1, nosuch proteins were observed in culture supernatant of strains expressingtruncated SLPs, except for P-1::pSL69 (data not shown for P-1::pSL70).These data suggest that, whereas the carboxy-terminal part of the SLPand in particular residues numbered 536 to 1252 in FIG. 6 aredispensable, the amino-terminal part and especially residues numbered 31to 269 in FIG. 6 are absolutely required for viability of P-1 cells.Residues numbered 31 to 269 in FIG. 6 constitute the N-terminal 239 or19.56% residues of the mature SLP.

Translational fusion of reporter proteins to the carboxy-terminus oftruncated SLPs. To determine whether the deletable part of SLP can bereplaced by a protein of interest, we fused reporter proteins NPTII andthe soluble fragment of the subunit S1 of toxin produced by Bordetellapertussis (PT) to the carboxy-terminus of truncated SLPs. To achievethis, a similar strategy as described above was used with themodification that the intermediate vectors now contained an internal slppart, translationally fused to either nptII (pSL102, pSL113; Table IV)or PT in fusion with NPTII (PSL111; Table IV). FIG. 17 summarises theexpected fusion proteins generated by the different intermediatevectors. Nm^(R) colonies, putative integrants, were selected afterintroduction of these intermediate vectors into P-1 by electroporationand selection of Em^(R) transformants. Southern hybridizations ofBglII-digested total DNA of these candidates with ³² P-labelled pSL20(containing a 4-kb BglII fragment of the slp gene; FIG. 15A), revealedthe expected patterns after single homologous recombination through thecloned slp part (FIG. 16).

In SDS-PAGE analysis of total protein extracts of recombinant strainsP-1::pSL69, P-1::pSL102 and P-1::pSL113 (FIG. 18A), major protein bandswere observed at 74 kDa, 102 kDa and 130 kDa, respectively, the sizesexpected for the truncated SLP or the two different fusion proteins. Intotal protein extracts of P-1::pSL111 (FIG. 18C), however, only a faintprotein band of the expected size (120 kDa) could be observed. Thefusion SLPs fractionated predominantly to the cell debris aftersonication, as was also observed for the truncated SLPs.

Western blottings of similar SDS-PAGE gels were challenged withanti-NPTII or anti-PT antibodies. Anti-NPTII reacted only with the102-kDa and 130-kDa proteins in P-1::pSL102 and P-1::pSL113 proteinextracts, respectively (FIG. 18C), whereas in P1::pSL111 extracts, the120-kDa protein was revealed (data not shown). No cross-reaction wasobserved with proteins from P-1::pSL69. Anti-PT detected two specificproteins in P-1::pSL111 (120 kDa and 90 kDa), and four specificlow-molecular mass proteins, that were also revealed in P-1::pSL69extracts (FIG. 18D). Signals with both anti-PT and anti-NPTII weresignificantly enhanced when using proteins from native sacculipreparations (see below).

Reporter proteins fused to SLP retain their enzymatic activity. Becauseboth proteins used as reporters in this study exhibit enzymatic activitythat can be relatively quickly assayed, we determined whether the fusionproteins retained these catalytic abilities.

Kanamycin phosphorylation activity of fusion proteins was determined bythe in gel assay, using either TCA-precipitated culture supernatants, orcell debris and soluble fraction after sonication. Significantphosphorylation activity was observed in all recombinant strains (butnot in P-1 or in P-1::pSL69 extracts) and was confirmed almostexclusively to the insoluble cell debris fraction after sonication (FIG.19).

NAD-glycohydrolase activity, specified by the S1 subunit of the B.pertussis toxin, was determined on TCA-precipitated culture supernatant,or cell debris and soluble fraction after sonication of the recombinantstrain P-1::pSL111 (Table V), in comparison to a calibration curve usingpurified PT toxin. Again, significant enzymatic activities were onlydetected in cellular debris fraction of P-1::pSL111. The apparentlyhigh, a specific hydrolase activity detected in the supernatant of bothP-1::pSL111 and P-1::pSL69 is due to acid hydrolysis caused by TCAresidues from the precipitation (data now shown).

                  TABLE V    ______________________________________    NAD-glycohydrolase activities of recombinant P-1 strains    Protein Source                 Released C14-nicotinamide (cmp)    ______________________________________    PT toxin (μg/ml)     0           1860     1           5350     5           10520    10           14810    20           21850    40           26050    P-1::pSL69    cell debris  1340    sonicate     1480    P-1::pSL111    cell debris  5380    sonicate     1530    ______________________________________

Carboxy-terminal fusions to SLP assemble in a functional S-layer. Toaddress the question whether fusion proteins between truncated SLP andNPTII assemble into a S-layer, intact bacteria were immunogold-labelledusing anti-NPTII antibodies. Significant accumulation of label on thebacteria was observed with P-1::pSL102 and to a lesser extent withP-1::pSL113 (FIGS. 20A and 20B). No background label could be foundusing either P-1 or P-1::pSL69 (which contains an intracellular NPTIIprotein).

Circumstantial evidence for the assembly of the fusion proteins into anS-layer, comes from the preparation of ghost cells, or native sacculi asdescribed by Sara and Sleytr (1987), in which cytoplasm and membrane areremoved without affecting the structural integrity of the peptide-glycanlayer and the S-layer. Application of this protocol to recombinantP-1::pSL102, and P-1::pSL111 strains resulted in sacculi preparations,significantly enriched in fusion S-layer protein (FIG. 21A). Westernblots, challenged with anti-NPTI or anti-PT detected readily the fusionproteins (FIGS. 21B and 21C).

The immunogenicity of the composite S-layers was determined by injectionof the recombinant bacteria P-1::pSL111, expressing the S1 subunit of PTfused to SLP, along with purified S1 and recombinant P-1::pSL102bacteria as controls. S1 antibody titers were determined after 12 weeksby ELISA (Table VI). A significant higher amount of S1-recognizingantibodies were detected in blood samples of the groups of mice injectedby the highest concentration of the recombinant bacteria expressing theS1-subunit in a composite S-layer (P-1::pSL111).

                  TABLE VI    ______________________________________    Immunogenicity of recombinant P-1::pSL111 in Balb/c mice            Antigen            (μg)   Dilution 1:20                                Dilution 1:80    ______________________________________    S1        0.1         19.7      14.9              1           426.1     402.1              10          465.5     460.8    S1 + AF   0.1         132.5     56.0              1           418.2     432.4              10          457.8     453.7    IB111     0.1         49.5      2.2              1           39.8      1.9              10          238.7     116.8    IB111 + AF              0.1         160.9     75.7              1           158.8     70.3              10          354.3     210.8    IB102     0.1         34.5      3.8              1           43.9      1.7              10          67.6      6.5    IB102 + AF              0.1         117.0     16.8              1           151.1     57.4              10          160.1     65.2    ______________________________________

Values are geometric means of ELISA titer readings of five independentinjections. AF, Freund's adjuvant; IB111, intact bacterial P-1::pSL111;IB102, intact bacterial P-1::pSL102.

                  TABLE VII    ______________________________________    Detailed information on the construction of intermediate    vectors for the construction of truncated SLP's and    composite SLP's.    Note on numbering: base 1 = A of ATG of the slp gene    (= mRNA numbering + 95)    Simple SLP clones    ______________________________________    pSL4  =     1536 bp HindIII fragment in pUC18 HindIII. This fragment                contains the complete slp promoter and slp gene portion 1                to 142.    pSL5  =     slp gene portion 49-807 (PvuII-PvuII) in pUC18 SmaI.    pSL10 =     slp gene portion 443-2053 (Bg1II-Bg1II) in                pUC18 BamHI.    pSL20 =     3048 bp HindIII fragment in pUC18 HindIII. This fragment                contains the slp gene portion 1049 to end of ORP (3759)                and the transcription termination signal.    ______________________________________    Truncated SLP intermediate vectors    Name   Cloned slp part                      Restriction site and clone                                     Site in pSL64    ______________________________________    pSL66  2048-2854  pSL20 Bg1II    BamHI    pSL68  1470-2476  pSL20 PvuII    EcoRV    pSL69  443-2053   pSL10 XbaI/EcoRI                                     XbaI/EcoRI    pSL70  443-1609   pSL10 XbaI/HpaI                                     XbaI/EcoRV    pSL71  49-807     pSL5 XbaI/EcoRI                                     XbaI/EcoRI    ______________________________________    Composite SLP intermediate vectors    ______________________________________    pSL109        554 bp Sau3A fragment cloned in BamHI site of                  pSL64 and selection of the correct orientation.    pSL102        EcoRI-Bg1III fragment of pSL20 (1049-2053) in                  BamHI site of pSL101.    pSL113        Bg1II fragment of pSL20 (2048-2854) in BamHI site                  of pSL101.    pSL111:       Same as pSL102 in BamHI site of pSL109.    pSL40   =     7453 bp high copy number bifunctional vector,                  Example 1 and FIG. 4A.    pSL84   =     6679 bp low-copy number bifuncfional vector,                  Example 1 and FIG. 4B.    pSL150  =     promoterless β-glucuronidase gene isolated as 2558 bp                  BamHI/SphI fragment and cloned in pSL40                  BamHI/SphI, Example 2.    pSL151-159            =     XbaI/NcoI fragments carrying progressive                  deletions of the slp promoter into pSL150                  XbaI/NcoI. Exact end points of the deletions are                  indicated in Example 2 and FIG. 10. NcoI site                  coincides with ATG start codon.    pSL64   =     1.12 kb BamHI/Sa1I fragment, carrying promoter-                  less nptII gene from pKm109/2 into pSL40                  BamHI/Sa1I, Example 3 and FIG. 14A.    pSL101  =     essentially the same as pSL64, but having another                  DNA linker in front of the nptII gene, Example 3 and                  FIG. 14B.    ______________________________________

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    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 25    (2) INFORMATION FOR SEQ ID NO: 1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:    TTCGGAAAAGATAGTGT17    (2) INFORMATION FOR SEQ ID NO: 2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 54 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:    CTAAATTTATGTCCCAATGCTTGAATTTCGGAAAAGATAGTGTTATATTATTGT54    (2) INFORMATION FOR SEQ ID NO: 3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 16 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:    ATTATTGAGAGTAAGG16    (2) INFORMATION FOR SEQ ID NO: 4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 45 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:    TCCAGAAAATGCTTGGTTATTATTGAGAGTAAGGTATAATAGGTA45    (2) INFORMATION FOR SEQ ID NO: 5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:    GTCTTTAATTTTTGACAA18    (2) INFORMATION FOR SEQ ID NO: 6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 44 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:    AAAATATTACGGGAGTCTTTAATTTTTGACAATTTAGTAACCAT44    (2) INFORMATION FOR SEQ ID NO: 7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 4197 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:    GAAAGCTATAATACATACATTTAGGTAACTAGGCGGTACTATAGTTTTCGTTGGATTAAT60    ATCAATTTAAGGAATTTTAGGGAGGAATACATTAATGGCAAAGCAAAACAAAGGCCGTAA120    GTTCTTCGCGGCATCAGCAACAGCTGCATTAGTTGCATCGGCAATCGTACCTGTAGCATC180    TGCTGCACAAGTAAACGACTATAACAAAATCTCTGGATACGCTAAAGAAGCAGTTCAAGC240    TTTAGTTGACCAAGGCGTAATCCAAGGTGATACTAACGGGAACTTCAACCCACTTAACAC300    AGTAACTCGTGCACAAGCTGCAGAAATCTTCACAAAAGCTTTAGAATTAGAAGCTAACGG360    AGATGTAAACTTCAAAGACGTGAAAGCTGGCGCTTGGTACTACAACTCAATCGCTGCTGT420    TGTAGCTAACGGCATTTTTGAAGGTGTTAGTGCAACTGAATTTGCACCAAACAAATCTTT480    AACTCGTTCTGAAGCTGCTAAAATTTTAGTAGAAGCATTCGGTTTAGAAGGTGAAGCAGA540    TCTTAGCGAATTTGCTGACGCTTCTCAAGTAAAACCTTGGGCTAAAAAATACTTAGAAAT600    CGCAGTAGCTAACGGCATTTTCGAAGGTACTGATGCAAACAAACTTAACCCTAACAACTC660    AATCACTCGTCAAGACTTTGCACTAGTGTTCAAACGTACAGTTGACAAAGTTGAAGGTGA720    AACTCCAGAAGAAGCAGCATTTGTTAAAGCTATCAACAACACAACTGTTGAAGTAACATT780    CGAAGAAGAAGTTACTAACGTTCAAGCACTTAACTTCAAAATCGAAGGTTTAGAAATTAA840    AAATGCTTCTGTTAAACAAACAAACAAAAAAGTTGTTGTATTAACTACTGAAGCTCAAAC900    AGCTGATAAAGAGTATGTTTTAACTCTTGACGGCGAAACAATCGGTGGCTTTAAAGGTGT960    GGCTGCTGTAGTTCCAACTAAAGTTGAACTAGTATCTTCTGCAGTTCAAGGTAAACTTGG1020    TCAAGAAGTAAAAGTTCAAGCTAAAGTAACTGTTGCTGAAGGTCAATCTAAAGCTGGTAT1080    TCCTGTTACTTTCACTGTACCAGGTAACAACAATGATGGCGTTGTACCAACATTAACAGG1140    TGAAGCTTTAACAAACGAAGAGGGTATCGCAACATACTCTTACACTCGTTATAAAGAAGG1200    TACTGATGAAGTAACTGCTTATGCAACTGGTGATCGTTCTAAATTCTCACTTGGTTATGT1260    ATTCTGGGGTGTAGATACAATTCTTTCAGTTGAAGAAGTAACTACAGGTGCTTCAGTTAA1320    TAATGGTGCAAACAAAACTTACAAAGTTACTTATAAAAACCCTAAAACTGGTAAACCAGA1380    AGCAAACAAAACATTTAATGTTGGTTTTGTAGAAAACATGAATGTTACTTCTGATAAAGT1440    AGCAAATGCTACAGTTAATGGCGTAAAAGCATTACAATTAAGCAATGGTACAGCTTTAGA1500    CGCTGCTCAAATTACAACAGATTCTAAAGGTGAAGCTACATTCACAGTTTCTGGTACTAA1560    TGCAGCTGTAACGCCAGTAGTATATGATCTACACAGCACTAACAATAGTACTTCAAATAA1620    AAAATATAGTGCATCTGCTTTACAAACTACTGCTTCTAAAGTAACTTTCGCTGCTCTTCA1680    AGCAGAGTATACAATTGAGTTAACTCGTGCTGATAATGCTGGAGAAGTTGCTGCAATTGG1740    CGCTACTAACGGTCGCGAATACAAAGTTATTGTAAAAGATAAAGCTGGTAACTTAGCTAA1800    AAATGAAATCGTTAATGTTGCATTCAATGAAGATAAAGATCGTGTAATTTCAACAGTTAC1860    AAATGCTAAATTCGTTGATACTGATCCAGATACTGCAGTATACTTCACAGGCGATAAAGC1920    AAAACAAATCTCTGTAAAAACAAATGATAAAGGTGAAGCTACATTTGTTATCGGTTCTGA1980    TACAGTAAACGATTATGCAACACCAATTGCTTGGATTGATATTAATACTTCTGATGCAAA2040    ACAAGGCGACCTTGATGAAGGTGAACCAAAAGCAGTTGCACCAATCTCTTACTTCCAAGC2100    ACCATATCTTGATGGCTCAGCTATCAAAGCATACAAAAAATCAGATCTTAATAAAGCTGT2160    AACTAAGTTTGATGGTTCTGAAACTGCAGTATTTGCAGCAGAATTAGTAAACCAAAGCGG2220    CAAAAAAGTAACTGGTACTTCTATTAAGAAAGCAACTTATACAATCTACAATACTGGTGC2280    TAATGATATTAAAGTAGATAACCAAGTTATCTCACCAAATCGTAGCTACACAGTAACTTA2340    TGAAGCTACTTTATCTTCTACAGGAACTGTTATTACACCTGCTAAGAATTTAGAAGTTAC2400    TTCAGTGGATGGTAAAACAACTGCTGTTAAAGTAATTGCTACAGGTATTGCTGTTAATAC2460    AGACGGTAAAGACTATGCATTTACTGCTAAAGAAGCTACAGCTACATTCACAGCTACAAA2520    TGAAGTTCCAAACTCTTACACTGGTGTAGCTACTCAATTCAATACAGCTGATTCTGGTTC2580    AAACAGCAACTCTATTTGGTTTGCTGGTAAAAACCCAGTGAAATATGCTGGTGTATCAGG2640    CAAAACATATAAATACTTCGGAGCTAATGGTAATGAAGTATTTGGTGAAGCGGCATGGGA2700    AGCATTATTAACTCAATATGCAACTGAAGGCCAAAAAGTAACAATCTCATATAATGTAGA2760    TGGTGATACAGTTACATTTAAAGTAATTAGTGCTGTTAATTCTTCAACTGAAGCTATCAA2820    ACCAGTTGCTCCAACAACACCAGCAGCTCCAACTACTGGCGCATTAACATTAACACCAGC2880    AGCTGGTGGTTTAGTTGATTTAACAACTGCAACTAACACTTTAGGAATTTCATTAGCTGA2940    TGCAGATCTTAATGTAAGTGCAACAACTGTTGATACTGCAACTGTTTCATTAAAAGATAG3000    TGCAAATAATTCATTATCTCTTACATTAGTTGAAACTGGTGCTAATACAGGTGTATTTGC3060    TACAACTGTTCAAGCTGGTACATTATCTTCTTTAACTGCTGGTACATTAACAGTTACTTA3120    TGCAGATGCTAAAAATGCTGCAGGTGTTGCTGAAAATATTACTGCTAGCGTAACATTAAA3180    GAAAACTACTGGAGCAATTACTTCTGATACATTTACACAAGGTGTATTACCATCAGCAGC3240    TACAGCAGCTGAATATACTTCTAAATCAATTGCTGCAGATTATACATTTGCAACAGGTGA3300    AGGATTCACTTTAAATATTGATAATGCTGGTGCTCAAGTAATTAACTTAGCAGGTAAAAA3360    AGGTGCACAAGGTGTAGCTGATGCTATCAATGCTACATTTGCAGGTACTGCAACTGTTTC3420    TGGAGACAAAGTAGTTATTAAATCAGCTACAACAGGTGTTGGTTCTGAAGTTGAAGTTAC3480    ATTCTCTTCTGTTAATCAAGTATTAAATGCAGTAGTTAACGGTAAAGATCAAGTCGTTGC3540    AGGAACAGCTGCTACAAAAGCATTCACGATTACTACAGCCCTTTCTGTGGGTGAAAAAGT3600    AGTTATTGATGGTGTTGAATATACTGCTGTAGCATTTGGAACTGCTCCAACAGCAAATAC3660    ATTCGTAGTTGAATCTGCTGCTAATACATTAGCTTCAGTAGCTGACCAAGCTGCAAATCT3720    TGCTGCTACAATTGATACTTTAAACACTGCAGATAAGTTTACAGCTTCTGCAACAGGTGC3780    TACTATTACATTAACTTCTACTGTAACACCAGTAGGTACTACAATTACTGAACCAGTAAT3840    TACATTAAAATAAGCAATTAACTTAAAATACTTTTAATTATTTGCCTATTTTATAATTTC3900    TATGACTCTATGAGATAACAATCTCATAGAGTCTTTTTTATTTTTAGAACCTCTAGATAG3960    AAAGAAATTTGAATTTATTATGAAATTTATAAAGAAGTCTTGTAACCTTTTATAAGGTAA4020    CTAGTCTAATTAAGAGAGTTATGTAAAAGCAATATATATCGATTCATATTATTTAAAAGG4080    CTAAAATTATTGTTTTAACTCAAACGGGGGTGGTAACAAAAGTTAATCAAGCAGCAATGA4140    GTTTTCTAGAAAATATTCATGAAATTCTGGAAATCCTTATTGCTTTATATGAAGCTT4197    (2) INFORMATION FOR SEQ ID NO: 8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 4197 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Bacillus sphaericus    (C) INDIVIDUAL ISOLATE: P-1    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION:95..3850    (ix) FEATURE:    (A) NAME/KEY: mat.sub.-- peptide    (B) LOCATION:185..3850    (ix) FEATURE:    (A) NAME/KEY: sig.sub.-- peptide    (B) LOCATION:95..184    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:    GAAAGCTATAATACATACATTTAGGTAACTAGGCGGTACTATAGTTTTCGTTGGATTAAT60    ATCAATTTAAGGAATTTTAGGGAGGAATACATTAATGGCAAAGCAAAACAAA112    MetAlaLysGlnAsnLys    30-25    GGCCGTAAGTTCTTCGCGGCATCAGCAACAGCTGCATTAGTTGCATCG160    GlyArgLysPhePheAlaAlaSerAlaThrAlaAlaLeuValAlaSer    20-15-10    GCAATCGTACCTGTAGCATCTGCTGCACAAGTAAACGACTATAACAAA208    AlaIleValProValAlaSerAlaAlaGlnValAsnAspTyrAsnLys    515    ATCTCTGGATACGCTAAAGAAGCAGTTCAAGCTTTAGTTGACCAAGGC256    IleSerGlyTyrAlaLysGluAlaValGlnAlaLeuValAspGlnGly    101520    GTAATCCAAGGTGATACTAACGGGAACTTCAACCCACTTAACACAGTA304    ValIleGlnGlyAspThrAsnGlyAsnPheAsnProLeuAsnThrVal    25303540    ACTCGTGCACAAGCTGCAGAAATCTTCACAAAAGCTTTAGAATTAGAA352    ThrArgAlaGlnAlaAlaGluIlePheThrLysAlaLeuGluLeuGlu    455055    GCTAACGGAGATGTAAACTTCAAAGACGTGAAAGCTGGCGCTTGGTAC400    AlaAsnGlyAspValAsnPheLysAspValLysAlaGlyAlaTrpTyr    606570    TACAACTCAATCGCTGCTGTTGTAGCTAACGGCATTTTTGAAGGTGTT448    TyrAsnSerIleAlaAlaValValAlaAsnGlyIlePheGluGlyVal    758085    AGTGCAACTGAATTTGCACCAAACAAATCTTTAACTCGTTCTGAAGCT496    SerAlaThrGluPheAlaProAsnLysSerLeuThrArgSerGluAla    9095100    GCTAAAATTTTAGTAGAAGCATTCGGTTTAGAAGGTGAAGCAGATCTT544    AlaLysIleLeuValGluAlaPheGlyLeuGluGlyGluAlaAspLeu    105110115120    AGCGAATTTGCTGACGCTTCTCAAGTAAAACCTTGGGCTAAAAAATAC592    SerGluPheAlaAspAlaSerGlnValLysProTrpAlaLysLysTyr    125130135    TTAGAAATCGCAGTAGCTAACGGCATTTTCGAAGGTACTGATGCAAAC640    LeuGluIleAlaValAlaAsnGlyIlePheGluGlyThrAspAlaAsn    140145150    AAACTTAACCCTAACAACTCAATCACTCGTCAAGACTTTGCACTAGTG688    LysLeuAsnProAsnAsnSerIleThrArgGlnAspPheAlaLeuVal    155160165    TTCAAACGTACAGTTGACAAAGTTGAAGGTGAAACTCCAGAAGAAGCA736    PheLysArgThrValAspLysValGluGlyGluThrProGluGluAla    170175180    GCATTTGTTAAAGCTATCAACAACACAACTGTTGAAGTAACATTCGAA784    AlaPheValLysAlaIleAsnAsnThrThrValGluValThrPheGlu    185190195200    GAAGAAGTTACTAACGTTCAAGCACTTAACTTCAAAATCGAAGGTTTA832    GluGluValThrAsnValGlnAlaLeuAsnPheLysIleGluGlyLeu    205210215    GAAATTAAAAATGCTTCTGTTAAACAAACAAACAAAAAAGTTGTTGTA880    GluIleLysAsnAlaSerValLysGlnThrAsnLysLysValValVal    220225230    TTAACTACTGAAGCTCAAACAGCTGATAAAGAGTATGTTTTAACTCTT928    LeuThrThrGluAlaGlnThrAlaAspLysGluTyrValLeuThrLeu    235240245    GACGGCGAAACAATCGGTGGCTTTAAAGGTGTGGCTGCTGTAGTTCCA976    AspGlyGluThrIleGlyGlyPheLysGlyValAlaAlaValValPro    250255260    ACTAAAGTTGAACTAGTATCTTCTGCAGTTCAAGGTAAACTTGGTCAA1024    ThrLysValGluLeuValSerSerAlaValGlnGlyLysLeuGlyGln    265270275280    GAAGTAAAAGTTCAAGCTAAAGTAACTGTTGCTGAAGGTCAATCTAAA1072    GluValLysValGlnAlaLysValThrValAlaGluGlyGlnSerLys    285290295    GCTGGTATTCCTGTTACTTTCACTGTACCAGGTAACAACAATGATGGC1120    AlaGlyIleProValThrPheThrValProGlyAsnAsnAsnAspGly    300305310    GTTGTACCAACATTAACAGGTGAAGCTTTAACAAACGAAGAGGGTATC1168    ValValProThrLeuThrGlyGluAlaLeuThrAsnGluGluGlyIle    315320325    GCAACATACTCTTACACTCGTTATAAAGAAGGTACTGATGAAGTAACT1216    AlaThrTyrSerTyrThrArgTyrLysGluGlyThrAspGluValThr    330335340    GCTTATGCAACTGGTGATCGTTCTAAATTCTCACTTGGTTATGTATTC1264    AlaTyrAlaThrGlyAspArgSerLysPheSerLeuGlyTyrValPhe    345350355360    TGGGGTGTAGATACAATTCTTTCAGTTGAAGAAGTAACTACAGGTGCT1312    TrpGlyValAspThrIleLeuSerValGluGluValThrThrGlyAla    365370375    TCAGTTAATAATGGTGCAAACAAAACTTACAAAGTTACTTATAAAAAC1360    SerValAsnAsnGlyAlaAsnLysThrTyrLysValThrTyrLysAsn    380385390    CCTAAAACTGGTAAACCAGAAGCAAACAAAACATTTAATGTTGGTTTT1408    ProLysThrGlyLysProGluAlaAsnLysThrPheAsnValGlyPhe    395400405    GTAGAAAACATGAATGTTACTTCTGATAAAGTAGCAAATGCTACAGTT1456    ValGluAsnMetAsnValThrSerAspLysValAlaAsnAlaThrVal    410415420    AATGGCGTAAAAGCATTACAATTAAGCAATGGTACAGCTTTAGACGCT1504    AsnGlyValLysAlaLeuGlnLeuSerAsnGlyThrAlaLeuAspAla    425430435440    GCTCAAATTACAACAGATTCTAAAGGTGAAGCTACATTCACAGTTTCT1552    AlaGlnIleThrThrAspSerLysGlyGluAlaThrPheThrValSer    445450455    GGTACTAATGCAGCTGTAACGCCAGTAGTATATGATCTACACAGCACT1600    GlyThrAsnAlaAlaValThrProValValTyrAspLeuHisSerThr    460465470    AACAATAGTACTTCAAATAAAAAATATAGTGCATCTGCTTTACAAACT1648    AsnAsnSerThrSerAsnLysLysTyrSerAlaSerAlaLeuGlnThr    475480485    ACTGCTTCTAAAGTAACTTTCGCTGCTCTTCAAGCAGAGTATACAATT1696    ThrAlaSerLysValThrPheAlaAlaLeuGlnAlaGluTyrThrIle    490495500    GAGTTAACTCGTGCTGATAATGCTGGAGAAGTTGCTGCAATTGGCGCT1744    GluLeuThrArgAlaAspAsnAlaGlyGluValAlaAlaIleGlyAla    505510515520    ACTAACGGTCGCGAATACAAAGTTATTGTAAAAGATAAAGCTGGTAAC1792    ThrAsnGlyArgGluTyrLysValIleValLysAspLysAlaGlyAsn    525530535    TTAGCTAAAAATGAAATCGTTAATGTTGCATTCAATGAAGATAAAGAT1840    LeuAlaLysAsnGluIleValAsnValAlaPheAsnGluAspLysAsp    540545550    CGTGTAATTTCAACAGTTACAAATGCTAAATTCGTTGATACTGATCCA1888    ArgValIleSerThrValThrAsnAlaLysPheValAspThrAspPro    555560565    GATACTGCAGTATACTTCACAGGCGATAAAGCAAAACAAATCTCTGTA1936    AspThrAlaValTyrPheThrGlyAspLysAlaLysGlnIleSerVal    570575580    AAAACAAATGATAAAGGTGAAGCTACATTTGTTATCGGTTCTGATACA1984    LysThrAsnAspLysGlyGluAlaThrPheValIleGlySerAspThr    585590595600    GTAAACGATTATGCAACACCAATTGCTTGGATTGATATTAATACTTCT2032    ValAsnAspTyrAlaThrProIleAlaTrpIleAspIleAsnThrSer    605610615    GATGCAAAACAAGGCGACCTTGATGAAGGTGAACCAAAAGCAGTTGCA2080    AspAlaLysGlnGlyAspLeuAspGluGlyGluProLysAlaValAla    620625630    CCAATCTCTTACTTCCAAGCACCATATCTTGATGGCTCAGCTATCAAA2128    ProIleSerTyrPheGlnAlaProTyrLeuAspGlySerAlaIleLys    635640645    GCATACAAAAAATCAGATCTTAATAAAGCTGTAACTAAGTTTGATGGT2176    AlaTyrLysLysSerAspLeuAsnLysAlaValThrLysPheAspGly    650655660    TCTGAAACTGCAGTATTTGCAGCAGAATTAGTAAACCAAAGCGGCAAA2224    SerGluThrAlaValPheAlaAlaGluLeuValAsnGlnSerGlyLys    665670675680    AAAGTAACTGGTACTTCTATTAAGAAAGCAACTTATACAATCTACAAT2272    LysValThrGlyThrSerIleLysLysAlaThrTyrThrIleTyrAsn    685690695    ACTGGTGCTAATGATATTAAAGTAGATAACCAAGTTATCTCACCAAAT2320    ThrGlyAlaAsnAspIleLysValAspAsnGlnValIleSerProAsn    700705710    CGTAGCTACACAGTAACTTATGAAGCTACTTTATCTTCTACAGGAACT2368    ArgSerTyrThrValThrTyrGluAlaThrLeuSerSerThrGlyThr    715720725    GTTATTACACCTGCTAAGAATTTAGAAGTTACTTCAGTGGATGGTAAA2416    ValIleThrProAlaLysAsnLeuGluValThrSerValAspGlyLys    730735740    ACAACTGCTGTTAAAGTAATTGCTACAGGTATTGCTGTTAATACAGAC2464    ThrThrAlaValLysValIleAlaThrGlyIleAlaValAsnThrAsp    745750755760    GGTAAAGACTATGCATTTACTGCTAAAGAAGCTACAGCTACATTCACA2512    GlyLysAspTyrAlaPheThrAlaLysGluAlaThrAlaThrPheThr    765770775    GCTACAAATGAAGTTCCAAACTCTTACACTGGTGTAGCTACTCAATTC2560    AlaThrAsnGluValProAsnSerTyrThrGlyValAlaThrGlnPhe    780785790    AATACAGCTGATTCTGGTTCAAACAGCAACTCTATTTGGTTTGCTGGT2608    AsnThrAlaAspSerGlySerAsnSerAsnSerIleTrpPheAlaGly    795800805    AAAAACCCAGTGAAATATGCTGGTGTATCAGGCAAAACATATAAATAC2656    LysAsnProValLysTyrAlaGlyValSerGlyLysThrTyrLysTyr    810815820    TTCGGAGCTAATGGTAATGAAGTATTTGGTGAAGCGGCATGGGAAGCA2704    PheGlyAlaAsnGlyAsnGluValPheGlyGluAlaAlaTrpGluAla    825830835840    TTATTAACTCAATATGCAACTGAAGGCCAAAAAGTAACAATCTCATAT2752    LeuLeuThrGlnTyrAlaThrGluGlyGlnLysValThrIleSerTyr    845850855    AATGTAGATGGTGATACAGTTACATTTAAAGTAATTAGTGCTGTTAAT2800    AsnValAspGlyAspThrValThrPheLysValIleSerAlaValAsn    860865870    TCTTCAACTGAAGCTATCAAACCAGTTGCTCCAACAACACCAGCAGCT2848    SerSerThrGluAlaIleLysProValAlaProThrThrProAlaAla    875880885    CCAACTACTGGCGCATTAACATTAACACCAGCAGCTGGTGGTTTAGTT2896    ProThrThrGlyAlaLeuThrLeuThrProAlaAlaGlyGlyLeuVal    890895900    GATTTAACAACTGCAACTAACACTTTAGGAATTTCATTAGCTGATGCA2944    AspLeuThrThrAlaThrAsnThrLeuGlyIleSerLeuAlaAspAla    905910915920    GATCTTAATGTAAGTGCAACAACTGTTGATACTGCAACTGTTTCATTA2992    AspLeuAsnValSerAlaThrThrValAspThrAlaThrValSerLeu    925930935    AAAGATAGTGCAAATAATTCATTATCTCTTACATTAGTTGAAACTGGT3040    LysAspSerAlaAsnAsnSerLeuSerLeuThrLeuValGluThrGly    940945950    GCTAATACAGGTGTATTTGCTACAACTGTTCAAGCTGGTACATTATCT3088    AlaAsnThrGlyValPheAlaThrThrValGlnAlaGlyThrLeuSer    955960965    TCTTTAACTGCTGGTACATTAACAGTTACTTATGCAGATGCTAAAAAT3136    SerLeuThrAlaGlyThrLeuThrValThrTyrAlaAspAlaLysAsn    970975980    GCTGCAGGTGTTGCTGAAAATATTACTGCTAGCGTAACATTAAAGAAA3184    AlaAlaGlyValAlaGluAsnIleThrAlaSerValThrLeuLysLys    9859909951000    ACTACTGGAGCAATTACTTCTGATACATTTACACAAGGTGTATTACCA3232    ThrThrGlyAlaIleThrSerAspThrPheThrGlnGlyValLeuPro    100510101015    TCAGCAGCTACAGCAGCTGAATATACTTCTAAATCAATTGCTGCAGAT3280    SerAlaAlaThrAlaAlaGluTyrThrSerLysSerIleAlaAlaAsp    102010251030    TATACATTTGCAACAGGTGAAGGATTCACTTTAAATATTGATAATGCT3328    TyrThrPheAlaThrGlyGluGlyPheThrLeuAsnIleAspAsnAla    103510401045    GGTGCTCAAGTAATTAACTTAGCAGGTAAAAAAGGTGCACAAGGTGTA3376    GlyAlaGlnValIleAsnLeuAlaGlyLysLysGlyAlaGlnGlyVal    105010551060    GCTGATGCTATCAATGCTACATTTGCAGGTACTGCAACTGTTTCTGGA3424    AlaAspAlaIleAsnAlaThrPheAlaGlyThrAlaThrValSerGly    1065107010751080    GACAAAGTAGTTATTAAATCAGCTACAACAGGTGTTGGTTCTGAAGTT3472    AspLysValValIleLysSerAlaThrThrGlyValGlySerGluVal    108510901095    GAAGTTACATTCTCTTCTGTTAATCAAGTATTAAATGCAGTAGTTAAC3520    GluValThrPheSerSerValAsnGlnValLeuAsnAlaValValAsn    110011051110    GGTAAAGATCAAGTCGTTGCAGGAACAGCTGCTACAAAAGCATTCACG3568    GlyLysAspGlnValValAlaGlyThrAlaAlaThrLysAlaPheThr    111511201125    ATTACTACAGCCCTTTCTGTGGGTGAAAAAGTAGTTATTGATGGTGTT3616    IleThrThrAlaLeuSerValGlyGluLysValValIleAspGlyVal    113011351140    GAATATACTGCTGTAGCATTTGGAACTGCTCCAACAGCAAATACATTC3664    GluTyrThrAlaValAlaPheGlyThrAlaProThrAlaAsnThrPhe    1145115011551160    GTAGTTGAATCTGCTGCTAATACATTAGCTTCAGTAGCTGACCAAGCT3712    ValValGluSerAlaAlaAsnThrLeuAlaSerValAlaAspGlnAla    116511701175    GCAAATCTTGCTGCTACAATTGATACTTTAAACACTGCAGATAAGTTT3760    AlaAsnLeuAlaAlaThrIleAspThrLeuAsnThrAlaAspLysPhe    118011851190    ACAGCTTCTGCAACAGGTGCTACTATTACATTAACTTCTACTGTAACA3808    ThrAlaSerAlaThrGlyAlaThrIleThrLeuThrSerThrValThr    119512001205    CCAGTAGGTACTACAATTACTGAACCAGTAATTACATTAAAA3850    ProValGlyThrThrIleThrGluProValIleThrLeuLys    121012151220    TAAGCAATTAACTTAAAATACTTTTAATTATTTGCCTATTTTATAATTTCTATGACTCTA3910    TGAGATAACAATCTCATAGAGTCTTTTTTATTTTTAGAACCTCTAGATAGAAAGAAATTT3970    GAATTTATTATGAAATTTATAAAGAAGTCTTGTAACCTTTTATAAGGTAACTAGTCTAAT4030    TAAGAGAGTTATGTAAAAGCAATATATATCGATTCATATTATTTAAAAGGCTAAAATTAT4090    TGTTTTAACTCAAACGGGGGTGGTAACAAAAGTTAATCAAGCAGCAATGAGTTTTCTAGA4150    AAATATTCATGAAATTCTGGAAATCCTTATTGCTTTATATGAAGCTT4197    (2) INFORMATION FOR SEQ ID NO: 9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1252 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:    MetAlaLysGlnAsnLysGlyArgLysPhePheAlaAlaSerAlaThr    30-25-20-15    AlaAlaLeuValAlaSerAlaIleValProValAlaSerAlaAlaGln    10-51    ValAsnAspTyrAsnLysIleSerGlyTyrAlaLysGluAlaValGln    51015    AlaLeuValAspGlnGlyValIleGlnGlyAspThrAsnGlyAsnPhe    202530    AsnProLeuAsnThrValThrArgAlaGlnAlaAlaGluIlePheThr    35404550    LysAlaLeuGluLeuGluAlaAsnGlyAspValAsnPheLysAspVal    556065    LysAlaGlyAlaTrpTyrTyrAsnSerIleAlaAlaValValAlaAsn    707580    GlyIlePheGluGlyValSerAlaThrGluPheAlaProAsnLysSer    859095    LeuThrArgSerGluAlaAlaLysIleLeuValGluAlaPheGlyLeu    100105110    GluGlyGluAlaAspLeuSerGluPheAlaAspAlaSerGlnValLys    115120125130    ProTrpAlaLysLysTyrLeuGluIleAlaValAlaAsnGlyIlePhe    135140145    GluGlyThrAspAlaAsnLysLeuAsnProAsnAsnSerIleThrArg    150155160    GlnAspPheAlaLeuValPheLysArgThrValAspLysValGluGly    165170175    GluThrProGluGluAlaAlaPheValLysAlaIleAsnAsnThrThr    180185190    ValGluValThrPheGluGluGluValThrAsnValGlnAlaLeuAsn    195200205210    PheLysIleGluGlyLeuGluIleLysAsnAlaSerValLysGlnThr    215220225    AsnLysLysValValValLeuThrThrGluAlaGlnThrAlaAspLys    230235240    GluTyrValLeuThrLeuAspGlyGluThrIleGlyGlyPheLysGly    245250255    ValAlaAlaValValProThrLysValGluLeuValSerSerAlaVal    260265270    GlnGlyLysLeuGlyGlnGluValLysValGlnAlaLysValThrVal    275280285290    AlaGluGlyGlnSerLysAlaGlyIleProValThrPheThrValPro    295300305    GlyAsnAsnAsnAspGlyValValProThrLeuThrGlyGluAlaLeu    310315320    ThrAsnGluGluGlyIleAlaThrTyrSerTyrThrArgTyrLysGlu    325330335    GlyThrAspGluValThrAlaTyrAlaThrGlyAspArgSerLysPhe    340345350    SerLeuGlyTyrValPheTrpGlyValAspThrIleLeuSerValGlu    355360365370    GluValThrThrGlyAlaSerValAsnAsnGlyAlaAsnLysThrTyr    375380385    LysValThrTyrLysAsnProLysThrGlyLysProGluAlaAsnLys    390395400    ThrPheAsnValGlyPheValGluAsnMetAsnValThrSerAspLys    405410415    ValAlaAsnAlaThrValAsnGlyValLysAlaLeuGlnLeuSerAsn    420425430    GlyThrAlaLeuAspAlaAlaGlnIleThrThrAspSerLysGlyGlu    435440445450    AlaThrPheThrValSerGlyThrAsnAlaAlaValThrProValVal    455460465    TyrAspLeuHisSerThrAsnAsnSerThrSerAsnLysLysTyrSer    470475480    AlaSerAlaLeuGlnThrThrAlaSerLysValThrPheAlaAlaLeu    485490495    GlnAlaGluTyrThrIleGluLeuThrArgAlaAspAsnAlaGlyGlu    500505510    ValAlaAlaIleGlyAlaThrAsnGlyArgGluTyrLysValIleVal    515520525530    LysAspLysAlaGlyAsnLeuAlaLysAsnGluIleValAsnValAla    535540545    PheAsnGluAspLysAspArgValIleSerThrValThrAsnAlaLys    550555560    PheValAspThrAspProAspThrAlaValTyrPheThrGlyAspLys    565570575    AlaLysGlnIleSerValLysThrAsnAspLysGlyGluAlaThrPhe    580585590    ValIleGlySerAspThrValAsnAspTyrAlaThrProIleAlaTrp    595600605610    IleAspIleAsnThrSerAspAlaLysGlnGlyAspLeuAspGluGly    615620625    GluProLysAlaValAlaProIleSerTyrPheGlnAlaProTyrLeu    630635640    AspGlySerAlaIleLysAlaTyrLysLysSerAspLeuAsnLysAla    645650655    ValThrLysPheAspGlySerGluThrAlaValPheAlaAlaGluLeu    660665670    ValAsnGlnSerGlyLysLysValThrGlyThrSerIleLysLysAla    675680685690    ThrTyrThrIleTyrAsnThrGlyAlaAsnAspIleLysValAspAsn    695700705    GlnValIleSerProAsnArgSerTyrThrValThrTyrGluAlaThr    710715720    LeuSerSerThrGlyThrValIleThrProAlaLysAsnLeuGluVal    725730735    ThrSerValAspGlyLysThrThrAlaValLysValIleAlaThrGly    740745750    IleAlaValAsnThrAspGlyLysAspTyrAlaPheThrAlaLysGlu    755760765770    AlaThrAlaThrPheThrAlaThrAsnGluValProAsnSerTyrThr    775780785    GlyValAlaThrGlnPheAsnThrAlaAspSerGlySerAsnSerAsn    790795800    SerIleTrpPheAlaGlyLysAsnProValLysTyrAlaGlyValSer    805810815    GlyLysThrTyrLysTyrPheGlyAlaAsnGlyAsnGluValPheGly    820825830    GluAlaAlaTrpGluAlaLeuLeuThrGlnTyrAlaThrGluGlyGln    835840845850    LysValThrIleSerTyrAsnValAspGlyAspThrValThrPheLys    855860865    ValIleSerAlaValAsnSerSerThrGluAlaIleLysProValAla    870875880    ProThrThrProAlaAlaProThrThrGlyAlaLeuThrLeuThrPro    885890895    AlaAlaGlyGlyLeuValAspLeuThrThrAlaThrAsnThrLeuGly    900905910    IleSerLeuAlaAspAlaAspLeuAsnValSerAlaThrThrValAsp    915920925930    ThrAlaThrValSerLeuLysAspSerAlaAsnAsnSerLeuSerLeu    935940945    ThrLeuValGluThrGlyAlaAsnThrGlyValPheAlaThrThrVal    950955960    GlnAlaGlyThrLeuSerSerLeuThrAlaGlyThrLeuThrValThr    965970975    TyrAlaAspAlaLysAsnAlaAlaGlyValAlaGluAsnIleThrAla    980985990    SerValThrLeuLysLysThrThrGlyAlaIleThrSerAspThrPhe    995100010051010    ThrGlnGlyValLeuProSerAlaAlaThrAlaAlaGluTyrThrSer    101510201025    LysSerIleAlaAlaAspTyrThrPheAlaThrGlyGluGlyPheThr    103010351040    LeuAsnIleAspAsnAlaGlyAlaGlnValIleAsnLeuAlaGlyLys    104510501055    LysGlyAlaGlnGlyValAlaAspAlaIleAsnAlaThrPheAlaGly    106010651070    ThrAlaThrValSerGlyAspLysValValIleLysSerAlaThrThr    1075108010851090    GlyValGlySerGluValGluValThrPheSerSerValAsnGlnVal    109511001105    LeuAsnAlaValValAsnGlyLysAspGlnValValAlaGlyThrAla    111011151120    AlaThrLysAlaPheThrIleThrThrAlaLeuSerValGlyGluLys    112511301135    ValValIleAspGlyValGluTyrThrAlaValAlaPheGlyThrAla    114011451150    ProThrAlaAsnThrPheValValGluSerAlaAlaAsnThrLeuAla    1155116011651170    SerValAlaAspGlnAlaAlaAsnLeuAlaAlaThrIleAspThrLeu    117511801185    AsnThrAlaAspLysPheThrAlaSerAlaThrGlyAlaThrIleThr    119011951200    LeuThrSerThrValThrProValGlyThrThrIleThrGluProVal    120512101215    IleThrLeuLys    1220    (2) INFORMATION FOR SEQ ID NO: 10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 90 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:    ATGGCAAAGCAAAACAAAGGCCGTAAGTTCTTCGCGGCATCAGCAACAGCTGCATTAGTT60    GCATCGGCAATCGTACCTGTAGCATCTGCT90    (2) INFORMATION FOR SEQ ID NO: 11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 90 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION:1..90    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:    ATGGCAAAGCAAAACAAAGGCCGTAAGTTCTTCGCGGCATCAGCAACA48    MetAlaLysGlnAsnLysGlyArgLysPhePheAlaAlaSerAlaThr    151015    GCTGCATTAGTTGCATCGGCAATCGTACCTGTAGCATCTGCT90    AlaAlaLeuValAlaSerAlaIleValProValAlaSerAla    202530    (2) INFORMATION FOR SEQ ID NO: 12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:    MetAlaLysGlnAsnLysGlyArgLysPhePheAlaAlaSerAlaThr    151015    AlaAlaLeuValAlaSerAlaIleValProValAlaSerAla    202530    (2) INFORMATION FOR SEQ ID NO: 13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 3666 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:    GCACAAGTAAACGACTATAACAAAATCTCTGGATACGCTAAAGAAGCAGTTCAAGCTTTA60    GTTGACCAAGGCGTAATCCAAGGTGATACTAACGGGAACTTCAACCCACTTAACACAGTA120    ACTCGTGCACAAGCTGCAGAAATCTTCACAAAAGCTTTAGAATTAGAAGCTAACGGAGAT180    GTAAACTTCAAAGACGTGAAAGCTGGCGCTTGGTACTACAACTCAATCGCTGCTGTTGTA240    GCTAACGGCATTTTTGAAGGTGTTAGTGCAACTGAATTTGCACCAAACAAATCTTTAACT300    CGTTCTGAAGCTGCTAAAATTTTAGTAGAAGCATTCGGTTTAGAAGGTGAAGCAGATCTT360    AGCGAATTTGCTGACGCTTCTCAAGTAAAACCTTGGGCTAAAAAATACTTAGAAATCGCA420    GTAGCTAACGGCATTTTCGAAGGTACTGATGCAAACAAACTTAACCCTAACAACTCAATC480    ACTCGTCAAGACTTTGCACTAGTGTTCAAACGTACAGTTGACAAAGTTGAAGGTGAAACT540    CCAGAAGAAGCAGCATTTGTTAAAGCTATCAACAACACAACTGTTGAAGTAACATTCGAA600    GAAGAAGTTACTAACGTTCAAGCACTTAACTTCAAAATCGAAGGTTTAGAAATTAAAAAT660    GCTTCTGTTAAACAAACAAACAAAAAAGTTGTTGTATTAACTACTGAAGCTCAAACAGCT720    GATAAAGAGTATGTTTTAACTCTTGACGGCGAAACAATCGGTGGCTTTAAAGGTGTGGCT780    GCTGTAGTTCCAACTAAAGTTGAACTAGTATCTTCTGCAGTTCAAGGTAAACTTGGTCAA840    GAAGTAAAAGTTCAAGCTAAAGTAACTGTTGCTGAAGGTCAATCTAAAGCTGGTATTCCT900    GTTACTTTCACTGTACCAGGTAACAACAATGATGGCGTTGTACCAACATTAACAGGTGAA960    GCTTTAACAAACGAAGAGGGTATCGCAACATACTCTTACACTCGTTATAAAGAAGGTACT1020    GATGAAGTAACTGCTTATGCAACTGGTGATCGTTCTAAATTCTCACTTGGTTATGTATTC1080    TGGGGTGTAGATACAATTCTTTCAGTTGAAGAAGTAACTACAGGTGCTTCAGTTAATAAT1140    GGTGCAAACAAAACTTACAAAGTTACTTATAAAAACCCTAAAACTGGTAAACCAGAAGCA1200    AACAAAACATTTAATGTTGGTTTTGTAGAAAACATGAATGTTACTTCTGATAAAGTAGCA1260    AATGCTACAGTTAATGGCGTAAAAGCATTACAATTAAGCAATGGTACAGCTTTAGACGCT1320    GCTCAAATTACAACAGATTCTAAAGGTGAAGCTACATTCACAGTTTCTGGTACTAATGCA1380    GCTGTAACGCCAGTAGTATATGATCTACACAGCACTAACAATAGTACTTCAAATAAAAAA1440    TATAGTGCATCTGCTTTACAAACTACTGCTTCTAAAGTAACTTTCGCTGCTCTTCAAGCA1500    GAGTATACAATTGAGTTAACTCGTGCTGATAATGCTGGAGAAGTTGCTGCAATTGGCGCT1560    ACTAACGGTCGCGAATACAAAGTTATTGTAAAAGATAAAGCTGGTAACTTAGCTAAAAAT1620    GAAATCGTTAATGTTGCATTCAATGAAGATAAAGATCGTGTAATTTCAACAGTTACAAAT1680    GCTAAATTCGTTGATACTGATCCAGATACTGCAGTATACTTCACAGGCGATAAAGCAAAA1740    CAAATCTCTGTAAAAACAAATGATAAAGGTGAAGCTACATTTGTTATCGGTTCTGATACA1800    GTAAACGATTATGCAACACCAATTGCTTGGATTGATATTAATACTTCTGATGCAAAACAA1860    GGCGACCTTGATGAAGGTGAACCAAAAGCAGTTGCACCAATCTCTTACTTCCAAGCACCA1920    TATCTTGATGGCTCAGCTATCAAAGCATACAAAAAATCAGATCTTAATAAAGCTGTAACT1980    AAGTTTGATGGTTCTGAAACTGCAGTATTTGCAGCAGAATTAGTAAACCAAAGCGGCAAA2040    AAAGTAACTGGTACTTCTATTAAGAAAGCAACTTATACAATCTACAATACTGGTGCTAAT2100    GATATTAAAGTAGATAACCAAGTTATCTCACCAAATCGTAGCTACACAGTAACTTATGAA2160    GCTACTTTATCTTCTACAGGAACTGTTATTACACCTGCTAAGAATTTAGAAGTTACTTCA2220    GTGGATGGTAAAACAACTGCTGTTAAAGTAATTGCTACAGGTATTGCTGTTAATACAGAC2280    GGTAAAGACTATGCATTTACTGCTAAAGAAGCTACAGCTACATTCACAGCTACAAATGAA2340    GTTCCAAACTCTTACACTGGTGTAGCTACTCAATTCAATACAGCTGATTCTGGTTCAAAC2400    AGCAACTCTATTTGGTTTGCTGGTAAAAACCCAGTGAAATATGCTGGTGTATCAGGCAAA2460    ACATATAAATACTTCGGAGCTAATGGTAATGAAGTATTTGGTGAAGCGGCATGGGAAGCA2520    TTATTAACTCAATATGCAACTGAAGGCCAAAAAGTAACAATCTCATATAATGTAGATGGT2580    GATACAGTTACATTTAAAGTAATTAGTGCTGTTAATTCTTCAACTGAAGCTATCAAACCA2640    GTTGCTCCAACAACACCAGCAGCTCCAACTACTGGCGCATTAACATTAACACCAGCAGCT2700    GGTGGTTTAGTTGATTTAACAACTGCAACTAACACTTTAGGAATTTCATTAGCTGATGCA2760    GATCTTAATGTAAGTGCAACAACTGTTGATACTGCAACTGTTTCATTAAAAGATAGTGCA2820    AATAATTCATTATCTCTTACATTAGTTGAAACTGGTGCTAATACAGGTGTATTTGCTACA2880    ACTGTTCAAGCTGGTACATTATCTTCTTTAACTGCTGGTACATTAACAGTTACTTATGCA2940    GATGCTAAAAATGCTGCAGGTGTTGCTGAAAATATTACTGCTAGCGTAACATTAAAGAAA3000    ACTACTGGAGCAATTACTTCTGATACATTTACACAAGGTGTATTACCATCAGCAGCTACA3060    GCAGCTGAATATACTTCTAAATCAATTGCTGCAGATTATACATTTGCAACAGGTGAAGGA3120    TTCACTTTAAATATTGATAATGCTGGTGCTCAAGTAATTAACTTAGCAGGTAAAAAAGGT3180    GCACAAGGTGTAGCTGATGCTATCAATGCTACATTTGCAGGTACTGCAACTGTTTCTGGA3240    GACAAAGTAGTTATTAAATCAGCTACAACAGGTGTTGGTTCTGAAGTTGAAGTTACATTC3300    TCTTCTGTTAATCAAGTATTAAATGCAGTAGTTAACGGTAAAGATCAAGTCGTTGCAGGA3360    ACAGCTGCTACAAAAGCATTCACGATTACTACAGCCCTTTCTGTGGGTGAAAAAGTAGTT3420    ATTGATGGTGTTGAATATACTGCTGTAGCATTTGGAACTGCTCCAACAGCAAATACATTC3480    GTAGTTGAATCTGCTGCTAATACATTAGCTTCAGTAGCTGACCAAGCTGCAAATCTTGCT3540    GCTACAATTGATACTTTAAACACTGCAGATAAGTTTACAGCTTCTGCAACAGGTGCTACT3600    ATTACATTAACTTCTACTGTAACACCAGTAGGTACTACAATTACTGAACCAGTAATTACA3660    TTAAAA3666    (2) INFORMATION FOR SEQ ID NO: 14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 3666 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION:1..3666    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:    GCACAAGTAAACGACTATAACAAAATCTCTGGATACGCTAAAGAAGCA48    AlaGlnValAsnAspTyrAsnLysIleSerGlyTyrAlaLysGluAla    151015    GTTCAAGCTTTAGTTGACCAAGGCGTAATCCAAGGTGATACTAACGGG96    ValGlnAlaLeuValAspGlnGlyValIleGlnGlyAspThrAsnGly    202530    AACTTCAACCCACTTAACACAGTAACTCGTGCACAAGCTGCAGAAATC144    AsnPheAsnProLeuAsnThrValThrArgAlaGlnAlaAlaGluIle    354045    TTCACAAAAGCTTTAGAATTAGAAGCTAACGGAGATGTAAACTTCAAA192    PheThrLysAlaLeuGluLeuGluAlaAsnGlyAspValAsnPheLys    505560    GACGTGAAAGCTGGCGCTTGGTACTACAACTCAATCGCTGCTGTTGTA240    AspValLysAlaGlyAlaTrpTyrTyrAsnSerIleAlaAlaValVal    65707580    GCTAACGGCATTTTTGAAGGTGTTAGTGCAACTGAATTTGCACCAAAC288    AlaAsnGlyIlePheGluGlyValSerAlaThrGluPheAlaProAsn    859095    AAATCTTTAACTCGTTCTGAAGCTGCTAAAATTTTAGTAGAAGCATTC336    LysSerLeuThrArgSerGluAlaAlaLysIleLeuValGluAlaPhe    100105110    GGTTTAGAAGGTGAAGCAGATCTTAGCGAATTTGCTGACGCTTCTCAA384    GlyLeuGluGlyGluAlaAspLeuSerGluPheAlaAspAlaSerGln    115120125    GTAAAACCTTGGGCTAAAAAATACTTAGAAATCGCAGTAGCTAACGGC432    ValLysProTrpAlaLysLysTyrLeuGluIleAlaValAlaAsnGly    130135140    ATTTTCGAAGGTACTGATGCAAACAAACTTAACCCTAACAACTCAATC480    IlePheGluGlyThrAspAlaAsnLysLeuAsnProAsnAsnSerIle    145150155160    ACTCGTCAAGACTTTGCACTAGTGTTCAAACGTACAGTTGACAAAGTT528    ThrArgGlnAspPheAlaLeuValPheLysArgThrValAspLysVal    165170175    GAAGGTGAAACTCCAGAAGAAGCAGCATTTGTTAAAGCTATCAACAAC576    GluGlyGluThrProGluGluAlaAlaPheValLysAlaIleAsnAsn    180185190    ACAACTGTTGAAGTAACATTCGAAGAAGAAGTTACTAACGTTCAAGCA624    ThrThrValGluValThrPheGluGluGluValThrAsnValGlnAla    195200205    CTTAACTTCAAAATCGAAGGTTTAGAAATTAAAAATGCTTCTGTTAAA672    LeuAsnPheLysIleGluGlyLeuGluIleLysAsnAlaSerValLys    210215220    CAAACAAACAAAAAAGTTGTTGTATTAACTACTGAAGCTCAAACAGCT720    GlnThrAsnLysLysValValValLeuThrThrGluAlaGlnThrAla    225230235240    GATAAAGAGTATGTTTTAACTCTTGACGGCGAAACAATCGGTGGCTTT768    AspLysGluTyrValLeuThrLeuAspGlyGluThrIleGlyGlyPhe    245250255    AAAGGTGTGGCTGCTGTAGTTCCAACTAAAGTTGAACTAGTATCTTCT816    LysGlyValAlaAlaValValProThrLysValGluLeuValSerSer    260265270    GCAGTTCAAGGTAAACTTGGTCAAGAAGTAAAAGTTCAAGCTAAAGTA864    AlaValGlnGlyLysLeuGlyGlnGluValLysValGlnAlaLysVal    275280285    ACTGTTGCTGAAGGTCAATCTAAAGCTGGTATTCCTGTTACTTTCACT912    ThrValAlaGluGlyGlnSerLysAlaGlyIleProValThrPheThr    290295300    GTACCAGGTAACAACAATGATGGCGTTGTACCAACATTAACAGGTGAA960    ValProGlyAsnAsnAsnAspGlyValValProThrLeuThrGlyGlu    315310315320    GCTTTAACAAACGAAGAGGGTATCGCAACATACTCTTACACTCGTTAT1008    AlaLeuThrAsnGluGluGlyIleAlaThrTyrSerTyrThrArgTyr    325330335    AAAGAAGGTACTGATGAAGTAACTGCTTATGCAACTGGTGATCGTTCT1056    LysGluGlyThrAspGluValThrAlaTyrAlaThrGlyAspArgSer    340345350    AAATTCTCACTTGGTTATGTATTCTGGGGTGTAGATACAATTCTTTCA1104    LysPheSerLeuGlyTyrValPheTrpGlyValAspThrIleLeuSer    355360365    GTTGAAGAAGTAACTACAGGTGCTTCAGTTAATAATGGTGCAAACAAA1152    ValGluGluValThrThrGlyAlaSerValAsnAsnGlyAlaAsnLys    370375380    ACTTACAAAGTTACTTATAAAAACCCTAAAACTGGTAAACCAGAAGCA1200    ThrTyrLysValThrTyrLysAsnProLysThrGlyLysProGluAla    385390395400    AACAAAACATTTAATGTTGGTTTTGTAGAAAACATGAATGTTACTTCT1248    AsnLysThrPheAsnValGlyPheValGluAsnMetAsnValThrSer    405410415    GATAAAGTAGCAAATGCTACAGTTAATGGCGTAAAAGCATTACAATTA1296    AspLysValAlaAsnAlaThrValAsnGlyValLysAlaLeuGlnLeu    420425430    AGCAATGGTACAGCTTTAGACGCTGCTCAAATTACAACAGATTCTAAA1344    SerAsnGlyThrAlaLeuAspAlaAlaGlnIleThrThrAspSerLys    435440445    GGTGAAGCTACATTCACAGTTTCTGGTACTAATGCAGCTGTAACGCCA1392    GlyGluAlaThrPheThrValSerGlyThrAsnAlaAlaValThrPro    450455460    GTAGTATATGATCTACACAGCACTAACAATAGTACTTCAAATAAAAAA1440    ValValTyrAspLeuHisSerThrAsnAsnSerThrSerAsnLysLys    465470475480    TATAGTGCATCTGCTTTACAAACTACTGCTTCTAAAGTAACTTTCGCT1488    TyrSerAlaSerAlaLeuGlnThrThrAlaSerLysValThrPheAla    485490495    GCTCTTCAAGCAGAGTATACAATTGAGTTAACTCGTGCTGATAATGCT1536    AlaLeuGlnAlaGluTyrThrIleGluLeuThrArgAlaAspAsnAla    500505510    GGAGAAGTTGCTGCAATTGGCGCTACTAACGGTCGCGAATACAAAGTT1584    GlyGluValAlaAlaIleGlyAlaThrAsnGlyArgGluTyrLysVal    515520525    ATTGTAAAAGATAAAGCTGGTAACTTAGCTAAAAATGAAATCGTTAAT1632    IleValLysAspLysAlaGlyAsnLeuAlaLysAsnGluIleValAsn    530535540    GTTGCATTCAATGAAGATAAAGATCGTGTAATTTCAACAGTTACAAAT1680    ValAlaPheAsnGluAspLysAspArgValIleSerThrValThrAsn    545550555560    GCTAAATTCGTTGATACTGATCCAGATACTGCAGTATACTTCACAGGC1728    AlaLysPheValAspThrAspProAspThrAlaValTyrPheThrGly    565570575    GATAAAGCAAAACAAATCTCTGTAAAAACAAATGATAAAGGTGAAGCT1776    AspLysAlaLysGlnIleSerValLysThrAsnAspLysGlyGluAla    580585590    ACATTTGTTATCGGTTCTGATACAGTAAACGATTATGCAACACCAATT1824    ThrPheValIleGlySerAspThrValAsnAspTyrAlaThrProIle    595600605    GCTTGGATTGATATTAATACTTCTGATGCAAAACAAGGCGACCTTGAT1872    AlaTrpIleAspIleAsnThrSerAspAlaLysGlnGlyAspLeuAsp    610615620    GAAGGTGAACCAAAAGCAGTTGCACCAATCTCTTACTTCCAAGCACCA1920    GluGlyGluProLysAlaValAlaProIleSerTyrPheGlnAlaPro    625630635640    TATCTTGATGGCTCAGCTATCAAAGCATACAAAAAATCAGATCTTAAT1968    TyrLeuAspGlySerAlaIleLysAlaTyrLysLysSerAspLeuAsn    645650655    AAAGCTGTAACTAAGTTTGATGGTTCTGAAACTGCAGTATTTGCAGCA2016    LysAlaValThrLysPheAspGlySerGluThrAlaValPheAlaAla    660665670    GAATTAGTAAACCAAAGCGGCAAAAAAGTAACTGGTACTTCTATTAAG2064    GluLeuValAsnGlnSerGlyLysLysValThrGlyThrSerIleLys    675680685    AAAGCAACTTATACAATCTACAATACTGGTGCTAATGATATTAAAGTA2112    LysAlaThrTyrThrIleTyrAsnThrGlyAlaAsnAspIleLysVal    690695700    GATAACCAAGTTATCTCACCAAATCGTAGCTACACAGTAACTTATGAA2160    AspAsnGlnValIleSerProAsnArgSerTyrThrValThrTyrGlu    705710715720    GCTACTTTATCTTCTACAGGAACTGTTATTACACCTGCTAAGAATTTA2208    AlaThrLeuSerSerThrGlyThrValIleThrProAlaLysAsnLeu    725730735    GAAGTTACTTCAGTGGATGGTAAAACAACTGCTGTTAAAGTAATTGCT2256    GluValThrSerValAspGlyLysThrThrAlaValLysValIleAla    740745750    ACAGGTATTGCTGTTAATACAGACGGTAAAGACTATGCATTTACTGCT2304    ThrGlyIleAlaValAsnThrAspGlyLysAspTyrAlaPheThrAla    755760765    AAAGAAGCTACAGCTACATTCACAGCTACAAATGAAGTTCCAAACTCT2352    LysGluAlaThrAlaThrPheThrAlaThrAsnGluValProAsnSer    770775780    TACACTGGTGTAGCTACTCAATTCAATACAGCTGATTCTGGTTCAAAC2400    TyrThrGlyValAlaThrGlnPheAsnThrAlaAspSerGlySerAsn    785790795800    AGCAACTCTATTTGGTTTGCTGGTAAAAACCCAGTGAAATATGCTGGT2448    SerAsnSerIleTrpPheAlaGlyLysAsnProValLysTyrAlaGly    805810815    GTATCAGGCAAAACATATAAATACTTCGGAGCTAATGGTAATGAAGTA2496    ValSerGlyLysThrTyrLysTyrPheGlyAlaAsnGlyAsnGluVal    820825830    TTTGGTGAAGCGGCATGGGAAGCATTATTAACTCAATATGCAACTGAA2544    PheGlyGluAlaAlaTrpGluAlaLeuLeuThrGlnTyrAlaThrGlu    835840845    GGCCAAAAAGTAACAATCTCATATAATGTAGATGGTGATACAGTTACA2592    GlyGlnLysValThrIleSerTyrAsnValAspGlyAspThrValThr    850855860    TTTAAAGTAATTAGTGCTGTTAATTCTTCAACTGAAGCTATCAAACCA2640    PheLysValIleSerAlaValAsnSerSerThrGluAlaIleLysPro    865870875880    GTTGCTCCAACAACACCAGCAGCTCCAACTACTGGCGCATTAACATTA2688    ValAlaProThrThrProAlaAlaProThrThrGlyAlaLeuThrLeu    885890895    ACACCAGCAGCTGGTGGTTTAGTTGATTTAACAACTGCAACTAACACT2736    ThrProAlaAlaGlyGlyLeuValAspLeuThrThrAlaThrAsnThr    900905910    TTAGGAATTTCATTAGCTGATGCAGATCTTAATGTAAGTGCAACAACT2784    LeuGlyIleSerLeuAlaAspAlaAspLeuAsnValSerAlaThrThr    915920925    GTTGATACTGCAACTGTTTCATTAAAAGATAGTGCAAATAATTCATTA2832    ValAspThrAlaThrValSerLeuLysAspSerAlaAsnAsnSerLeu    930935940    TCTCTTACATTAGTTGAAACTGGTGCTAATACAGGTGTATTTGCTACA2880    SerLeuThrLeuValGluThrGlyAlaAsnThrGlyValPheAlaThr    945950955960    ACTGTTCAAGCTGGTACATTATCTTCTTTAACTGCTGGTACATTAACA2928    ThrValGlnAlaGlyThrLeuSerSerLeuThrAlaGlyThrLeuThr    965970975    GTTACTTATGCAGATGCTAAAAATGCTGCAGGTGTTGCTGAAAATATT2976    ValThrTyrAlaAspAlaLysAsnAlaAlaGlyValAlaGluAsnIle    980985990    ACTGCTAGCGTAACATTAAAGAAAACTACTGGAGCAATTACTTCTGAT3024    ThrAlaSerValThrLeuLysLysThrThrGlyAlaIleThrSerAsp    99510001005    ACATTTACACAAGGTGTATTACCATCAGCAGCTACAGCAGCTGAATAT3072    ThrPheThrGlnGlyValLeuProSerAlaAlaThrAlaAlaGluTyr    101010151020    ACTTCTAAATCAATTGCTGCAGATTATACATTTGCAACAGGTGAAGGA3120    ThrSerLysSerIleAlaAlaAspTyrThrPheAlaThrGlyGluGly    1025103010351040    TTCACTTTAAATATTGATAATGCTGGTGCTCAAGTAATTAACTTAGCA3168    PheThrLeuAsnIleAspAsnAlaGlyAlaGlnValIleAsnLeuAla    104510501055    GGTAAAAAAGGTGCACAAGGTGTAGCTGATGCTATCAATGCTACATTT3216    GlyLysLysGlyAlaGlnGlyValAlaAspAlaIleAsnAlaThrPhe    106010651070    GCAGGTACTGCAACTGTTTCTGGAGACAAAGTAGTTATTAAATCAGCT3264    AlaGlyThrAlaThrValSerGlyAspLysValValIleLysSerAla    107510801085    ACAACAGGTGTTGGTTCTGAAGTTGAAGTTACATTCTCTTCTGTTAAT3312    ThrThrGlyValGlySerGluValGluValThrPheSerSerValAsn    109010951100    CAAGTATTAAATGCAGTAGTTAACGGTAAAGATCAAGTCGTTGCAGGA3360    GlnValLeuAsnAlaValValAsnGlyLysAspGlnValValAlaGly    1105111011151120    ACAGCTGCTACAAAAGCATTCACGATTACTACAGCCCTTTCTGTGGGT3408    ThrAlaAlaThrLysAlaPheThrIleThrThrAlaLeuSerValGly    112511301135    GAAAAAGTAGTTATTGATGGTGTTGAATATACTGCTGTAGCATTTGGA3456    GluLysValValIleAspGlyValGluTyrThrAlaValAlaPheGly    114011451150    ACTGCTCCAACAGCAAATACATTCGTAGTTGAATCTGCTGCTAATACA3504    ThrAlaProThrAlaAsnThrPheValValGluSerAlaAlaAsnThr    115511601165    TTAGCTTCAGTAGCTGACCAAGCTGCAAATCTTGCTGCTACAATTGAT3552    LeuAlaSerValAlaAspGlnAlaAlaAsnLeuAlaAlaThrIleAsp    117011751180    ACTTTAAACACTGCAGATAAGTTTACAGCTTCTGCAACAGGTGCTACT3600    ThrLeuAsnThrAlaAspLysPheThrAlaSerAlaThrGlyAlaThr    1185119011951200    ATTACATTAACTTCTACTGTAACACCAGTAGGTACTACAATTACTGAA3648    IleThrLeuThrSerThrValThrProValGlyThrThrIleThrGlu    120512101215    CCAGTAATTACATTAAAA3666    ProValIleThrLeuLys    1220    (2) INFORMATION FOR SEQ ID NO: 15:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1222 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:    AlaGlnValAsnAspTyrAsnLysIleSerGlyTyrAlaLysGluAla    151015    ValGlnAlaLeuValAspGlnGlyValIleGlnGlyAspThrAsnGly    202530    AsnPheAsnProLeuAsnThrValThrArgAlaGlnAlaAlaGluIle    354045    PheThrLysAlaLeuGluLeuGluAlaAsnGlyAspValAsnPheLys    505560    AspValLysAlaGlyAlaTrpTyrTyrAsnSerIleAlaAlaValVal    65707580    AlaAsnGlyIlePheGluGlyValSerAlaThrGluPheAlaProAsn    859095    LysSerLeuThrArgSerGluAlaAlaLysIleLeuValGluAlaPhe    100105110    GlyLeuGluGlyGluAlaAspLeuSerGluPheAlaAspAlaSerGln    115120125    ValLysProTrpAlaLysLysTyrLeuGluIleAlaValAlaAsnGly    130135140    IlePheGluGlyThrAspAlaAsnLysLeuAsnProAsnAsnSerIle    145150155160    ThrArgGlnAspPheAlaLeuValPheLysArgThrValAspLysVal    165170175    GluGlyGluThrProGluGluAlaAlaPheValLysAlaIleAsnAsn    180185190    ThrThrValGluValThrPheGluGluGluValThrAsnValGlnAla    195200205    LeuAsnPheLysIleGluGlyLeuGluIleLysAsnAlaSerValLys    210215220    GlnThrAsnLysLysValValValLeuThrThrGluAlaGlnThrAla    225230235240    AspLysGluTyrValLeuThrLeuAspGlyGluThrIleGlyGlyPhe    245250255    LysGlyValAlaAlaValValProThrLysValGluLeuValSerSer    260265270    AlaValGlnGlyLysLeuGlyGlnGluValLysValGlnAlaLysVal    275280285    ThrValAlaGluGlyGlnSerLysAlaGlyIleProValThrPheThr    290295300    ValProGlyAsnAsnAsnAspGlyValValProThrLeuThrGlyGlu    305310315320    AlaLeuThrAsnGluGluGlyIleAlaThrTyrSerTyrThrArgTyr    325330335    LysGluGlyThrAspGluValThrAlaTyrAlaThrGlyAspArgSer    340345350    LysPheSerLeuGlyTyrValPheTrpGlyValAspThrIleLeuSer    355360365    ValGluGluValThrThrGlyAlaSerValAsnAsnGlyAlaAsnLys    370375380    ThrTyrLysValThrTyrLysAsnProLysThrGlyLysProGluAla    385390395400    AsnLysThrPheAsnValGlyPheValGluAsnMetAsnValThrSer    405410415    AspLysValAlaAsnAlaThrValAsnGlyValLysAlaLeuGlnLeu    420425430    SerAsnGlyThrAlaLeuAspAlaAlaGlnIleThrThrAspSerLys    435440445    GlyGluAlaThrPheThrValSerGlyThrAsnAlaAlaValThrPro    450455460    ValValTyrAspLeuHisSerThrAsnAsnSerThrSerAsnLysLys    465470475480    TyrSerAlaSerAlaLeuGlnThrThrAlaSerLysValThrPheAla    485490495    AlaLeuGlnAlaGluTyrThrIleGluLeuThrArgAlaAspAsnAla    500505510    GlyGluValAlaAlaIleGlyAlaThrAsnGlyArgGluTyrLysVal    515520525    IleValLysAspLysAlaGlyAsnLeuAlaLysAsnGluIleValAsn    530535540    ValAlaPheAsnGluAspLysAspArgValIleSerThrValThrAsn    545550555560    AlaLysPheValAspThrAspProAspThrAlaValTyrPheThrGly    565570575    AspLysAlaLysGlnIleSerValLysThrAsnAspLysGlyGluAla    580585590    ThrPheValIleGlySerAspThrValAsnAspTyrAlaThrProIle    595600605    AlaTrpIleAspIleAsnThrSerAspAlaLysGlnGlyAspLeuAsp    610615620    GluGlyGluProLysAlaValAlaProIleSerTyrPheGlnAlaPro    625630635640    TyrLeuAspGlySerAlaIleLysAlaTyrLysLysSerAspLeuAsn    645650655    LysAlaValThrLysPheAspGlySerGluThrAlaValPheAlaAla    660665670    GluLeuValAsnGlnSerGlyLysLysValThrGlyThrSerIleLys    675680685    LysAlaThrTyrThrIleTyrAsnThrGlyAlaAsnAspIleLysVal    690695700    AspAsnGlnValIleSerProAsnArgSerTyrThrValThrTyrGlu    705710715720    AlaThrLeuSerSerThrGlyThrValIleThrProAlaLysAsnLeu    725730735    GluValThrSerValAspGlyLysThrThrAlaValLysValIleAla    740745750    ThrGlyIleAlaValAsnThrAspGlyLysAspTyrAlaPheThrAla    755760765    LysGluAlaThrAlaThrPheThrAlaThrAsnGluValProAsnSer    770775780    TyrThrGlyValAlaThrGlnPheAsnThrAlaAspSerGlySerAsn    785790795800    SerAsnSerIleTrpPheAlaGlyLysAsnProValLysTyrAlaGly    805810815    ValSerGlyLysThrTyrLysTyrPheGlyAlaAsnGlyAsnGluVal    820825830    PheGlyGluAlaAlaTrpGluAlaLeuLeuThrGlnTyrAlaThrGlu    835840845    GlyGlnLysValThrIleSerTyrAsnValAspGlyAspThrValThr    850855860    PheLysValIleSerAlaValAsnSerSerThrGluAlaIleLysPro    865870875880    ValAlaProThrThrProAlaAlaProThrThrGlyAlaLeuThrLeu    885890895    ThrProAlaAlaGlyGlyLeuValAspLeuThrThrAlaThrAsnThr    900905910    LeuGlyIleSerLeuAlaAspAlaAspLeuAsnValSerAlaThrThr    915920925    ValAspThrAlaThrValSerLeuLysAspSerAlaAsnAsnSerLeu    930935940    SerLeuThrLeuValGluThrGlyAlaAsnThrGlyValPheAlaThr    945950955960    ThrValGlnAlaGlyThrLeuSerSerLeuThrAlaGlyThrLeuThr    965970975    ValThrTyrAlaAspAlaLysAsnAlaAlaGlyValAlaGluAsnIle    980985990    ThrAlaSerValThrLeuLysLysThrThrGlyAlaIleThrSerAsp    99510001005    ThrPheThrGlnGlyValLeuProSerAlaAlaThrAlaAlaGluTyr    101010151020    ThrSerLysSerIleAlaAlaAspTyrThrPheAlaThrGlyGluGly    1025103010351040    PheThrLeuAsnIleAspAsnAlaGlyAlaGlnValIleAsnLeuAla    104510501055    GlyLysLysGlyAlaGlnGlyValAlaAspAlaIleAsnAlaThrPhe    106010651070    AlaGlyThrAlaThrValSerGlyAspLysValValIleLysSerAla    107510801085    ThrThrGlyValGlySerGluValGluValThrPheSerSerValAsn    109010951100    GlnValLeuAsnAlaValValAsnGlyLysAspGlnValValAlaGly    1105111011151120    ThrAlaAlaThrLysAlaPheThrIleThrThrAlaLeuSerValGly    112511301135    GluLysValValIleAspGlyValGluTyrThrAlaValAlaPheGly    114011451150    ThrAlaProThrAlaAsnThrPheValValGluSerAlaAlaAsnThr    115511601165    LeuAlaSerValAlaAspGlnAlaAlaAsnLeuAlaAlaThrIleAsp    117011751180    ThrLeuAsnThrAlaAspLysPheThrAlaSerAlaThrGlyAlaThr    1185119011951200    IleThrLeuThrSerThrValThrProValGlyThrThrIleThrGlu    120512101215    ProValIleThrLeuLys    1220    (2) INFORMATION FOR SEQ ID NO: 16:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 63 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (v) FRAGMENT TYPE: N-terminal    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Bacillus sphaericus    (C) INDIVIDUAL ISOLATE: P-1    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:    GCACAAGTAAACGACTATAACAAAATCTCTGGATACGCTAAAGAAGCAGTTCAAGCTTTA60    GTT63    (2) INFORMATION FOR SEQ ID NO: 17:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 63 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (v) FRAGMENT TYPE: N-terminal    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Bacillus sphaericus    (C) INDIVIDUAL ISOLATE: P-1    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION:1..63    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:    GCACAAGTAAACGACTATAACAAAATCTCTGGATACGCTAAAGAAGCA48    AlaGlnValAsnAspTyrAsnLysIleSerGlyTyrAlaLysGluAla    151015    GTTCAAGCTTTAGTT63    ValGlnAlaLeuVal    20    (2) INFORMATION FOR SEQ ID NO: 18:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:    AlaGlnValAsnAspTyrAsnLysIleSerGlyTyrAlaLysGluAla    151015    ValGlnAlaLeuVal    20    (2) INFORMATION FOR SEQ ID NO: 19:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 198 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE:    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:    MetAlaLysGlnAsnLysGlyArgLysPhePheAlaAlaSerAlaThr    151015    AlaAlaLeuValAlaSerAlaIleValProValAlaSerAlaAlaGln    202530    ValAsnAspTyrAsnLysIleSerGlyTyrAlaLysGluAlaValGln    354045    AlaLeuValAspGlnGlyValIleGlnGlyAspThrAsnGlyAsnPhe    505560    AsnProLeuAsnThrValThrArgAlaGlnAlaAlaGluIlePheThr    65707580    LysAlaLeuGluLeuGluAlaAsnGlyAspValAsnPheLysAspVal    859095    LysAlaGlyAlaTrpTyrTyrAsnSerIleAlaAlaValValAlaAsn    100105110    GlyIlePheGluGlyValSerAlaThrGluPheAlaProAsnLysSer    115120125    LeuThrArgSerGluAlaAlaLysIleLeuValGluAlaPheGlyLeu    130135140    GluGlyGluAlaAspLeuSerGluPheAlaAspAlaSerGlnValLys    145150155160    ProTrpAlaLysLysTyrLeuGluIleAlaValAlaAsnGlyIlePhe    165170175    GluGlyThrAspAlaAsnLysLeuAsnProAsnAsnSerIleThrArg    180185190    GlnAspPheAlaLeuVal    195    (2) INFORMATION FOR SEQ ID NO: 20:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 200 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE:    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:    MetAlaLysGlnAsnLysGlyArgLysPhePheAlaAlaSerAlaThr    151015    AlaAlaLeuValAlaSerAlaIleValProValAlaSerAlaAlaGln    202530    LeuMetAspPheAsnLysIleSerGlyTyrAlaLysGluAlaValGln    354045    SerLeuValAspAlaGlyValIleGlnGlyAspAlaAsnGlyAsnPhe    505560    AsnProLeuLysThrIleSerArgAlaGluAlaAlaThrIlePheThr    65707580    AsnAlaLeuGluLeuGluAlaGluGlyAspValAsnPheLysAspVal    859095    LysAlaAspAlaTrpTyrTyrAspAlaIleAlaAlaThrValGluAsn    100105110    GlyIlePheGluGlyValSerAlaThrGluPheAlaProAsnLysGln    115120125    LeuThrArgSerGluAlaAlaLysIleLeuValAspAlaPheGluLeu    130135140    GluGlyGluGlyAspLeuSerGluPheAlaAspAlaSerThrValLys    145150155160    ProTrpAlaLysSerTyrLeuGluIleAlaValAlaAsnGlyValIle    165170175    LysGlySerGluAlaAsnGlyLysThrAsnLeuAsnProAsnAlaPro    180185190    IleThrArgGlnAspPheAlaVal    195200    (2) INFORMATION FOR SEQ ID NO: 21:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 600 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:    GAATTCGCTAAGAAACGCCTTCTATATTTCGGTTTCTTTACAATTATAACTAAAATATTA60    CGGGAGTCTTTAATTTTTGACAATTTAGTAACCATTCCAGAAAATGCTTGGTTATTATTG120    AGAGTAAGGTATAATAGGTAACGGAACTATATGTTACCAATCCAAATGAGGATATAATTA180    GTTGTAATTTTAATGGTTTCTACCAAATACCATATTAGGTATGGTAAAAAAATCTTCTAT240    AACTAAATTTATGTCCCAATGCTTGAATTTCGGAAAAGATAGTGTTATATTATTGTAGAA300    AGTGAATAAACTTACTAGAATGGTATTCTACTACGCTTTTTCTAGTAAATTTACTAACAA360    ATTTGCTTTAGTTTTGTATTATTCAAGAAAGCTATAATACATACATTTAGGTAACTAGGC420    GGTACTATAGTTTTCGTTGGATTAATATCAATTTAAGGAATTTTAGGGAGGAATACATTA480    ATGGCAAAGCAAAACAAAGGCCGTAAGTTCTTCGCGGCATCAGCAACAGCTGCATTAGTT540    GCATCGGCAATCGTACCTGTAGCATCTGCTGCACAAGTAAACGACTATAACAAAATCTCT600    (2) INFORMATION FOR SEQ ID NO: 22:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 120 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:    ATGGCAAAGCAAAACAAAGGCCGTAAGTTCTTCGCGGCATCAGCAACAGCTGCATTAGTT60    GCATCGGCAATCGTACCTGTAGCATCTGCTGCACAAGTAAACGACTATAACAAAATCTCT120    (2) INFORMATION FOR SEQ ID NO: 23:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 84 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:    TCTAGAGGTACCGCATGCGATATCGAGCTCTCCCGGGAATTCCCGGGGATCCGGCCCATG60    ATCATGTGGATTGAACAAGATGGA84    (2) INFORMATION FOR SEQ ID NO: 24:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 71 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:    TCTAGAGGTACCGCATGCGATATCGAGCTCTCCCGGGAATTCCCGGGGATCCCTCGAGGA60    GCTTCGATGCA71    (2) INFORMATION FOR SEQ ID NO: 25:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (A) DESCRIPTION: /desc ="(synthetic    oligodeoxynucleotide)"    (ix) FEATURE:    (A) NAME/KEY: modified.sub.-- base    (B) LOCATION:6    (ix) FEATURE:    (A) NAME/KEY: modified.sub.-- base    (B) LOCATION:9    (ix) FEATURE:    (A) NAME/KEY: modified.sub.-- base    (B) LOCATION:18    (ix) FEATURE:    (A) NAME/KEY: modified.sub.-- base    (B) LOCATION:21    (ix) FEATURE:    (A) NAME/KEY: modified.sub.-- .sub.-- .sub.-- se    (B) LOCATION:24    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:    GCYTGNACNGCYTCYTTNGCNTANCC26    __________________________________________________________________________

We claim:
 1. A process of transforming B. sphaericus P-1 cells with DNA,which process comprises harvesting B. sphaericus P-1 cells at the latestationary growth phase, mixing the harvested cells with the DNA andeffecting electroporation to cause entry of the DNA into the said cells.